Biomineralization is a well-known phenomenon in plants and animals (Skinner and Jahren, 2003). In plants, biomineralization is usually found at the level of individual cells, either in epidermal cell walls or as intracellular structures such as cystoliths. Plant biomineralization shows surprising structural and compositional diversity. Calcium oxalate is generally found as intracellular biomineral whereas calcium carbonate is both found as intracellular cystoliths and in the cell walls of trichomes and stinging hairs (Franceschi and Horner, 1980; Lanning and Eleuterius, 1989; He et al., 2014). Silica is the hardest biomineral and widely reported in the form of deposits in the outer cell walls of the epidermis and trichomes, but also as intracellular phytoliths (Nawaz et al., 2019; Gallaher et al., 2020). Abrasive grainy cell wall inclusions and mineralized sharp tips of trichomes, as well as a dense cover of stiff mineralized hairs forming a physical barrier, are assumed to provide protection against grazing animals (Figure 1). The diversity of structural plant biominerals has recently been expanded by the discovery of calcium phosphate-based structures in plants (Ensikat et al., 2016; Ensikat et al., 2017; Weigend et al., 2018). Our SEM studies on the complex plant trichomes of Loasaceae (“Rock nettles,” Cornales) revealed the presence of calcium phosphate in high concentrations at specific sites in the cell wall, such as trichome tips, while the bulk of the trichome wall is typically mineralized with calcium carbonate. Additional studies have demonstrated its wide distribution across a range of plant families including Urticaceae, Boraginaceae, and Brassicaceae since the first discovery of calcium phosphate as a structural plant biomineral in Loasaceae (Mustafa et al., 2018a; Mustafa et al., 2018b; Weigend et al., 2018). Loasaceae are known for morphologically remarkably diverse trichome cover consisting of stinging hairs, much smaller barbed glochidiate trichomes, and scabrid trichomes with sharp tips (Weigend, 2003; Figure 2). Light microscopic (LM) images of Loasaceae trichomes (Figures 2B–G) illustrate the thickness of the walls and whether the trichomes are hollow or massive. Stinging hairs (Figure 2B) are essentially hollow, whereas the small glochidiate and scabrid trichomes are initially hollow, but later become massive (Figures 2C–G). Similar filling of small trichomes occurs in numerous species, such as Urtica dioica and Galium aparine (Figures 2H,I).

Detailed ontogenetic studies demonstrated that mineralization is initiated at the apex and in the trichome hooks and proceeds towards the trichome base; younger, partially mineralized trichomes still may have a non-mineralized base (Mustafa et al., 2017).

The three biominerals reported in plant trichomes are also found in the animal kingdom. Calcium phosphate and calcium carbonate, and to a lesser degree silica, play a major role in medicine, such as in bone formation. This and other applications are the basis for a rapidly growing body of studies on biomimetic materials based on apatite (calcium phosphate) (e.g., Vandecandelaere et al., 2012; Cazalbou et al., 2015; Haider et al., 2017; Nesseri et al., 2020; Bertolotti et al., 2021), calcium carbonate (Sommerdijk and de With, 2008; Wu and Wang 2013) and silica (Knecht and Wright, 2003; Jackson et al., 2015; Piletska et al., 2017). Despite this broad range of studies, very few focus on the exact localization, formation, composition and properties of plant biominerals. Trichome biomineralization is particularly useful for functional and physiological studies because the process of biomineralization can be studied more or less directly with suitable techniques. Previous studies examined the trichome surfaces for their elemental composition with various SEM-techniques, but we have little knowledge of the chemical composition of the cell walls below the surface, even though this likely plays a major role in functionality. Transmission electron microscopic (TEM) studies of thin sections and light microscopy of stained sections revealed the stratification of silicified and calcified cell wall structures of stinging hairs, and the different composition of stinging hair walls in Loasaceae, Euphorbiaceae, and Urticaceae have been known for a long time (Thurston, 1969; Thurston and Lersten, 1969), but the analytical capabilities in TEM were very limited at that time. Recently, Hughes et al. (2017) studied mineral deposits and stratification of biominerals in Urtica stinging hairs, while recently published detailed analyses of element distribution in stinging hairs, particularly of the genus Nasa, demonstrate complex deposition patterns of silica and calcium phosphate in trichome apices (Mustafa et al., 2017; Mustafa et al., 2018b). Raman spectroscopy as an analytical method with light microscopy resolution provides compositional data about organic and inorganic components of the samples with the possibility of analysing living trichomes, if they are kept immersed in water to avoid heat problems (Ensikat and Weigend, 2019).

We focus on species with triple biomineralization, i.e., containing SiO₂, CaCO₃, and Ca-phosphate (Ensikat et al., 2017) including the differential distribution of organic substances. Patterns of biomineralization at the micro-scale are investigated by sectioning and SEM block-face imaging of embedded samples. Complementary data from Raman spectroscopy are provided to understand both the inorganic compounds in trichome walls and their associated organic matrix substances. Our study strives to advance our knowledge of plant biomineralization and provide the basis for further exploration with sophisticated and high-resolution techniques and the tools of molecular physiology.

Scanning electron microscopy (SEM) with integrated energy-dispersive X-ray spectroscopy (EDX) for element analysis is an excellent tool for identification of biominerals. However, surface analyses provide only an incomplete picture when mineralization extends into the volume of the sample. In such cases, sections or transmission views with LM, including Raman spectroscopy, are suitable to comprehensively characterize the sample composition.

Loasaceae trichomes have previously been shown to display complex patterns of biomineralization (Ensikat et al., 2017). The present study is an extension of these studies to provide a three-dimensional view of biomineralization and a first look at the organic phase in inorganic/organic composites within trichome cells. The most striking result is the extreme zonation of mineral deposition across the cell walls and wall protuberances (barbs), with different minerals deposited with pinpoint precision. Similar complexity of cell wall structures are known from non-mineralized plant tissues. Plant cell walls are, unlike membranes of animal tissues, prominent structures contributing to the mechanical stability. Different cell wall composition, including hydrophilic carbohydrates (mainly cellulose) and hydrophobic components such as lignin, cutin and waxes, determine the permeability for water and ions. Tracheids or the cells of the Casparian strip are two examples, where the functional divergence in different wall portions is mediated by different types and quantities of organic compounds. Our observation of differentially mineralized trichome walls expands this phenomenon to the outer plant surface, where chemical heterogeneity at the micrometre level is expressed with the presence of up to three different mineral components in combination with different organic compounds.

The mineralization patterns in plant trichomes appear to reflect a range of functions, including plant defence. The tips and hooks of the trichomes are rendered particularly hard and sharp by the inclusion of either silica or calcium phosphate, enabling them to damage skin, mucous membranes and/or the exoskeleton of insects and other herbivores. Conversely, stinging hairs retain a certain degree of brittleness, so that the apex can break off and the caustic stinging substances injected into the attacker from the hollow interior of the mineralized trichome cell, functioning as hypodermic syringe (Thurston and Lersten, 1969; Tuberville et al., 1996). In trichomes containing both calcium carbonate and calcium phosphate there tends to be a trend towards phosphate “coatings,” i.e., the part of the trichome exposed to the elements is “varnished” with calcium phosphate, possibly to make use of its lower water solubility, as shown by the leaching experiments. Defensive trichomes in other plant species may be functionally very similar to those of Loasaceae without being mineralized: Stinging hairs of Cnidoscolus are morphologically very similar to those of Loasaceae or Urticaceae, but are entirely made up of organic polymers (Mustafa et al., 2018b). Overall, biomineralization of the cell wall shows extraordinary diversity: Different parts of the cell wall are incrusted with different mineral components (calcium carbonate, calcium phosphate, silica), in different structural contexts and relative concentrations, with varying amounts of an organic matrix. The distal and apical parts, where “rigidity” is of paramount importance, are nearly completely mineralized with minute organic admixtures. Other parts of the trichome, especially the shaft, have a higher proportion of cellulose and/or pectin, as shown by Raman spectroscopy, likely creating a composite material with higher strength and flexibility. In addition to the biomineralization of wall structures, we also find an amorphous “filling” of calcium carbonate in glochidiate trichomes, progressing basiscopically with trichome age. Both the amorphous filling and the calcium carbonate mineralization of the cell walls may be easily mobilized upon contact with water. Solubility of calcium-based biominerals in water has long been known as a problem in preparations for TEM investigations. It has been reported that during the ultramicrotomy of bone tissue sections floating on a water surface lose part of their mineral content; dry sectioning is required to avoid this artefact (Boothroyd, 1964; Thorogood and Gray, 1975; Studer et al., 2011).

Crystallinity is not necessarily an attribute of biominerals. Biogenic silica, for example, is generally amorphous. Biogenic plant calcium carbonate has been found as crystalline calcite in the seed coat (pericarp) of Lithospermum (Hilger et al., 1993; own data, not shown), whereas analyses of trichomes by Raman spectroscopy and X-ray powder diffraction always indicates amorphous material. Admixtures of various additives, such as phosphoproteins or inorganic phosphate, may stabilise amorphous CaCO₃ (Bentov et al., 2010; Gal et al., 2010).

The biomineral silica widely occurs as nearly pure substance in very hard structures or surface coatings. Thus, its integration into the wall of glochidiate trichome shafts of Blumenbachia is a remarkable novelty. In several species of other plant families, e.g., Urtica mairei and Urera baccifera, cell walls of trichomes are mainly mineralized with calcium phosphate instead of carbonate (Mustafa et al., 2018b). Thus, all three biominerals calcium carbonate, calcium phosphate, and silica can occur in concentrated form (in trichome tips or fillings in the cell lumen) or as composites with cellulose and pectin in the cell walls. Biomineralization in plants, particularly the formation of calcium carbonate and phosphate, has received little attention in the past, even though calcium phosphate biomineralization is of considerable interest in medicine and for comparatives studies of the process of biomineralization in animals such as vertebrates (Dorozhkin and Epple, 2002; Dillon et al., 2019). Exploration of the physicochemical properties of biominerals is a promising field of research for biomedical applications (Al-Kattan et al., 2012; Credou and Berthelot, 2015; Haider et al., 2017; Konovalova et al., 2017; Markov et al., 2017; Hickey and Pelling, 2019; Nesseri et al., 2020; Tschon et al., 2020) and may get a novel stimulus by the expansion into carbohydrate-based biomineral composites.

Recent research focusses on the chemical and physical properties of plant biominerals and the physiological pathways leading to their formation (Kumar et al., 2021). The accessibility of plant trichomes for in vivo studies and analysis by light microscopy and Raman spectroscopy renders them highly suitable subjects for such studies.