Hans-Jürgen Ensikat

Direct Access to Plant Epicuticular Wax Crystals by a New Mechanical Isolation Method.

A new method for the isolation of wax crystals from plant surfaces is presented. The wax‐covered plant surface, e.g., a piece of a leaf or fruit, is brought into contact with a preparation liquid, e.g., glycerol or triethylene glycol, and cooled to ca. −100°C. When the plant specimen is removed, the epicuticular wax remains embedded in the frozen liquid. After it warms up, the wax layer can be captured on appropriate carriers for further studies. This isolation method causes very little stress on the wax crystals; thus the shape and crystal structure are well preserved. In many cases it is possible, by choosing a preparation liquid with appropriate wettability, to isolate either the entire epicuticular wax layer or only discrete wax crystals without the underlying wax film. These crystals are well suited for electron diffraction studies by transmission electron microscopy and high resolution imaging by atomic force microscopy. The absence of intracuticular components and other impurities and the feasibility of the selective isolation of wax crystals enable improved chemical analysis and a more detailed study of their properties.

Liquid substitution: A versatile procedure for SEM specimen preparation of biological materials without drying or coating

Certain liquids with a very low vapour pressure, such as glycerol or triethylene glycol, can be used to infiltrate biological specimens so that they may be observed in the scanning electron microscope (SEM) without drying. The conductive properties of the fluids allow specimens to be examined either uncoated or with very thin coatings. The advantages of liquid substitution include the retention of lipids, waxes, loose particles, and surface contaminants. Since the procedure does not require expensive equipment, it offers an alternative to critical point drying or cryo‐preparation. For certain types of specimens, liquid substitution may represent the best preparation procedure. In addition, the fluids themselves may be imaged directly in the SEM, or indirectly by cathodoluminescence following labelling with fluorochromes.

Self-healing of voids in the wax coating on plant surfaces

The cuticles of plants provide a multifunctional interface between the plants and their environments. The cuticle, with its associated waxes, is a protective layer that minimizes water loss by transpiration and provides several functions, such as hydrophobicity, light reflection and absorption of harmful radiation. The self-healing of voids in the epicuticular wax layer has been studied in 17 living plants by atomic force microscopy (AFM), and the process of wax film formation is described. Two modes of wax film formation, a concentric layer formation and striped layer formation, were found, and the process of multilayer wax film formation is discussed. A new method for the preparation of small pieces of fresh, water-containing plant specimens for AFM investigations is introduced. The technique allows AFM investigations of several hours duration without significant shrinkage or lateral drift of the specimen. This research shows how plants refill voids in their surface wax layers by wax self-assembly and should be useful for the design of self-healing materials.

Diversity of calcium oxalate crystals in Cactaceae

The occurrence of various types of calcium oxalate crystals was studied in 251 species and subspecies of Cactaceae to determine whether they are useful characters for Cactaceae systematics. Crystal...

A first report of hydroxylated apatite as structural biomineral in Loasaceae – plants’ teeth against herbivores

Biomineralization provides living organisms with various materials for the formation of resilient structures. Calcium phosphate is the main component of teeth and bones in vertebrates, whereas especially silica serves for the protection against herbivores on many plant surfaces. Functional calcium phosphate structures are well-known from the animal kingdom, but had not so far been reported from higher plants. Here, we document the occurrence of calcium phosphate biomineralization in the South-American plant group Loasaceae (rock nettle family), which have stinging trichomes similar to those of the well-known stinging nettles (Urtica). Stinging hairs and the smaller, glochidiate trichomes contained nanocrystalline hydroxylated apatite, especially in their distal portions, replacing the silica found in analogous structures of other flowering plants. This could be demonstrated by chemical, spectroscopic, and diffraction analyses. Some species of Loasaceae contained both calcium phosphate and silica in addition to calcium carbonate. The intriguing discovery of structural hydroxylated apatite in plants invites further studies, e.g., on its systematic distribution across the family, the genetic and cellular control of plant biomineralization, the properties and ultrastructure of calcium phosphate. It may prove the starting point for the development of biomimetic calcium phosphate composites based on a cellulose matrix.

Fossil nutlets of Boraginaceae from the continental Eocene of Hamada of Méridja (southwestern Algeria): The first fossil of the Borage family in Africa

PREMISE OF THE STUDY The Paleogene deposits of the Hamada of Méridja, southwestern Algeria, are currently dated as lower-to-middle Eocene in age based on fossil gastropods and charophytes. Here we report the presence of fruits that can be assigned to the Boraginaceae s.str., apparently representing the first fossil record for this family in Africa, shedding new light on the historical biogeography of this group. METHODS Microscopic studies of the fossil nutlets were carried out and compared to extant Boraginaceae nutlets, and to types reported in the literature for this family. KEY RESULTS The fossils are strikingly similar in general size and morphology, particularly in the finer details of the attachment scar and ornamentation, to nutlets of extant representatives of the Boraginaceae tribe Echiochileae, and especially the genus Ogastemma. We believe that these nutlets represent an extinct member of this lineage. CONCLUSIONS The Ogastemma-like fossils indicate that the Echiochileae, which are most diverse in northern Africa and southwestern Asia, have a long history in this region, dating back to the Eocene. This tribe corresponds to the basal-most clade in Boraginaceae s.str., and the fossils described here agree well with an assumed African origin of the family and the Boraginales I, providing an important additional calibration point for dating the phylogenies of this clade.

Ontogeny and the process of biomineralization in the trichomes of Loasaceae

PREMISE OF THE STUDY South American Loasaceae have a morphologically complex trichome cover, which is characterized by multiple biomineralization. The current study investigates the ontogeny of these complex trichomes and the process of their biomineralization, since both are very poorly understood. METHODS The development of stinging trichomes on various parts of the plants and the process of mineralization were studied using scanning electron microscopy (SEM) and energy-dispersive x-ray spectroscopy (EDX). KEY RESULTS Trichomes are initiated very early in organ development and the different trichome types begin developing their distinctive morphology at a very early developmental stage. Biomineralization in the stinging trichomes starts with the deposition of silica or calcium phosphate in the apex and then proceeds basipetally, with a more irregular, subsimultaneous mineralization of the base and the shaft. Mineralization of the scabrid-glochidiate trichomes starts on the surface processes and in the apex (silica, calcium phosphate), with a subsequent mineralization of the shaft with calcium carbonate. CONCLUSION Mineralized trichomes in Loasaceae provide an excellent model for the study of biomineralization. The overall sequence of mineralization is typically from distal and peripheral to proximal and central. Typically, three biominerals-silica, calcium carbonate, and calcium phosphate-are differentially and sequentially deposited in different parts of each unicellular stinging trichome.

Complex patterns of multiple biomineralization in single-celled plant trichomes of the Loasaceae

PREMISE OF THE STUDY Plants of the family Loasaceae are characterized by a usually dense indument of various trichome types, including two basically different types of mineralized, unicellular trichomes (stinging hairs or setae and scabrid-glochidiate trichomes). Mineralized trichomes have long been known to have silicified or calcified walls, but recent studies demonstrated that trichomes of Loasaceae may also contain calcium phosphate. The current study investigates the distribution of different biominerals in the mineralized trichomes across several different taxa. METHODS Plants from cultivation were studied with scanning electron microscopy including energy dispersive x-ray analyses and element mapping. KEY RESULTS The vast majority of the 31 species investigated had at least two different biominerals in their trichomes, and 22 had three different biominerals in their trichomes. Thirty of the species had calcium phosphate in their trichomes. Loasa was mostly free of silica, but contained calcium phosphate in trichome tips and barbs, whereas calcium phosphate and silica were found in representatives of other genera of the family (Blumenbachia, Caiophora, Nasa). CONCLUSIONS Biomineralization is remarkably diversified between species, different trichome types and parts of the same trichome. Individual genera largely had different patterns of biomineralization. The presence of three biominerals in the trichomes of the basally branching Eucnide urens indicates either an early evolution and subsequent loss or several independent origins of multiple biomineralization. Differential biomineralization of the parts of individual, unicellular trichomes clearly indicates an extraordinary degree of physiological control over this process.

Calcium phosphate in plant trichomes: the overlooked biomineral

AbstractMain conclusionCalcium phosphate was unknown as a plant biomineral until recently reported in Neotropical Loasaceae. Here, we demonstrate its widespread occurrence in the trichomes of several plant families, including Brassicaceae. Calcium phosphate is the primary biomineral in, e.g., the bones and teeth of higher animals; in plants, it was only recently discovered in the stinging hairs and scabrid–glochidiate trichomes of South American Loasaceae (Ensikat et al. in Sci Rep UK 6:26073, 2016), where it appears to be deposited highly specifically, often replacing the common plant biomineral silica. We initiated a broader survey in a range of different plant orders to investigate a possibly wider distribution of calcium phosphate biomineralization in plants. Scanning electron microscopy with EDX element analysis and mapping was used for the detection of the biominerals: calcium phosphate, calcium carbonate, and silica in the trichomes of several common plant species of different orders. Results were authenticated with Raman spectroscopy. Calcium phosphate was found in the trichomes of several species in the orders Malpighiales, Rosales, Boraginales, and Brassicales. It occurred in trichome tips, replacing the more common silica, or together with silica and calcium carbonate at specific locations in the trichome cell walls. Most surprisingly, it was found in the trichomes of Arabidopsis thaliana, one of the most studied plant species—where it had been overlooked so far. The wide distribution of calcium phosphate as plant biomineral here demonstrated and the striking mineralization patterns with three different biominerals in the walls of single-celled trichomes underscore an unexpected complexity in plant biomineralization.

Mineralized trichomes in Boraginales: complex microscale heterogeneity and simple phylogenetic patterns

Background and Aims Boraginales are often characterized by a dense cover of stiff, mineralized trichomes, which may act as a first line of defence against herbivores. Recent studies have demonstrated that the widely reported silica and calcium carbonate in plant trichomes may be replaced by calcium phosphate. The present study investigates mineralization patterns in 42 species from nine families of the order Boraginales to investigate detailed patterns of mineralization and the possible presence of a phylogenetic signal in different mineralization patterns. Methods The distribution of biominerals was analysed by scanning electron microscopy (SEM) including cryo-SEM and energy-dispersive X-ray analyses with element mapping. The observed distribution of biominerals was plotted onto a published phylogeny of the Boraginales. Three colours were selected to represent the principal elements: Si (red), Ca (green) and P (blue). Key Results Calcium carbonate was present in the mineralized trichomes of all 42 species investigated, silica in 30 and calcium phosphate in 25; multiple mineralization with calcium carbonate and silica or calcium phosphate was found in all species, and 13 of the species were mineralized with all three biominerals. Trichome tips featured the most regular pattern - nearly all were exclusively mineralized with either silica or calcium phosphate. Biomineralization of the trichome shafts and bases was found to be more variable between species. However, the trichome bases were also frequently mineralized with calcium phosphate or silica, indicating that not only the tip is under functional constraints requiring specific patterns of chemical heterogeneity. The complete absence of either silica or phosphate may be an additional feature with systematic relevance. Conclusions This study demonstrates that complex, site-specific and differential biomineralization is widespread across the order Boraginales. Calcium phosphate, only recently first reported as a structural plant biomineral, is common and appears to be functionally analogous to silica. A comparison with the phylogeny of Boraginales additionally reveals striking phylogenetic patterns. Most families show characteristic patterns of biomineralization, such as the virtual absence of calcium phosphate in Cordiaceae and Boraginaceae, the triple biomineralization of Heliotropiaceae and Ehretiaceae, or the absence of silica in Namaceae and Codonaceae. The complex chemical and phylogenetic patterns indicate that trichome evolution and functionalities are anything but simple and follow complex functional and phylogenetic constraints.