Zygospore development of Spirogyra (Charophyta) investigated by serial block-face scanning electron microscopy and 3D reconstructions.

Sexual reproduction of Zygnematophyceae by conjugation is a less investigated topic due to the difficulties of the induction of this process and zygospore ripening under laboratory conditions. For this study, we collected field sampled zygospores of Spirogyra mirabilis and three additional Spirogyra strains in Austria and Greece. Serial block-face scanning electron microscopy was performed on high pressure frozen and freeze substituted zygospores and 3D reconstructions were generated, allowing a comprehensive insight into the process of zygospore maturation, involving storage compound and organelle rearrangements. Chloroplasts are drastically changed, while young stages contain both parental chloroplasts, the male chloroplasts are aborted and reorganised as 'secondary vacuoles' which initially contain plastoglobules and remnants of thylakoid membranes. The originally large pyrenoids and the volume of starch granules is significantly reduced during maturation (young: 8 ± 5 µm³, mature: 0.2 ± 0.2 µm³). In contrast, lipid droplets (LDs) increase significantly in number upon zygospore maturation, while simultaneously getting smaller (young: 21 ± 18 µm³, mature: 0.1 ± 0.2 and 0.5 ± 0.9 µm³). Only in S. mirabilis the LD volume increases (34 ± 29 µm³), occupying ~50% of the zygospore volume. Mature zygospores contain barite crystals as confirmed by Raman spectroscopy with a size of 0.02 - 0.05 µm³. The initially thin zygospore cell wall (~0.5 µm endospore, ~0.8 µm exospore) increases in thickness and develops a distinct, electron dense mesospore, which has a reticulate appearance (~1.4 µm) in Spirogyra sp. from Greece. The exo- and endospore show cellulose microfibrils in a helicoidal pattern. In the denser endospore, pitch angles of the microfibril layers were calculated: ~18 ± 3° in S. mirabilis, ~20 ± 3° in Spirogyra sp. from Austria and ~38 ± 8° in Spirogyra sp. from Greece. Overall this study gives new insights into Spirogyra sp. zygospore development, crucial for survival during dry periods and dispersal of this genus.

Hydrolyzable tannins are incorporated into the endocarp during sclerification of the water caltrop Trapa natans.

The water caltrop (Trapa natans) develops unique woody fruits with unusually large seeds among aquatic plants. During fruit development, the inner fruit wall (endocarp) sclerifies and forms a protective layer for the seed. Endocarp sclerification also occurs in many land plants with large seeds; however, in T. natans, the processes of fruit formation, endocarp hardening, and seed storage take place entirely underwater. To identify potential chemical and structural adaptations for the aquatic environment, we investigated the cell-wall composition in the endocarp at a young developmental stage, as well as at fruit maturity. Our work shows that hydrolyzable tannins-specifically gallotannins-flood the endocarp tissue during secondary wall formation and are integrated into cell walls along with lignin during maturation. Within the secondary walls of mature tissue, we identified unusually strong spectroscopic features of ester linkages, suggesting that the gallotannins and their derivatives are cross-linked to other wall components via ester bonds, leading to unique cell-wall properties. The synthesis of large amounts of water-soluble, defensive aromatic metabolites during secondary wall formation might be a fast way to defend seeds within the insufficiently lignified endocarp of T. natans.

The walnut shell network: 3D visualisation of symplastic and apoplastic transport routes in sclerenchyma tissue.

MAIN CONCLUSION: High symplastic connectivity via pits was linked to the lignification of the developing walnut shell. With maturation, this network lessened, whereas apoplastic intercellular space remained and became relevant for shell drying. The shell of the walnut (Juglans regia) sclerifies within several weeks. This fast secondary cell wall thickening and lignification of the shell tissue might need metabolites from the supporting husk tissue. To reveal the transport capacity of the walnut shell tissue and its connection to the husk, we visualised the symplastic and apoplastic transport routes during shell development by serial block face-SEM and 3D reconstruction. We found an extensive network of pit channels connecting the cells within the shell tissue, but even more towards the husk tissue. Each pit channel ended in a pit field, which was occupied by multiple plasmodesmata passing through the middle lamella. During shell development, secondary cell wall formation progressed towards the interior of the cell, leaving active pit channels open. In contrast, pit channels, which had no plasmodesmata connection to a neighbouring cell, got filled by cellulose layers from the inner cell wall lamellae. A comparison with other nut species showed that an extended network during sclerification seemed to be linked to high cell wall lignification and that the connectivity between cells got reduced with maturation. In contrast, intercellular spaces between cells remained unchanged during the entire sclerification process, allowing air and water to flow through the walnut shell tissue when mature. The connectivity between inner tissue and environment was essential during shell drying in the last month of nut development to avoid mould formation. The findings highlight how connectivity and transport work in developing walnut shell tissue and how finally in the mature state these structures influence shell mechanics, permeability, conservation and germination.

Twist and lock: nutshell structures for high strength and energy absorption.

Nutshells achieve remarkable properties by optimizing structure and chemistry at different hierarchical levels. Probing nutshells from the cellular down to the nano- and molecular level by microchemical and nanomechanical imaging techniques reveals insights into nature's packing concepts. In walnut and pistachio shells, carbohydrate and lignin polymers assemble to form thick-walled puzzle cells, which interlock three-dimensionally and show high tissue strength. Pistachio additionally achieves high-energy absorption by numerous lobes interconnected via ball-joint-like structures. By contrast, the three times more lignified walnut shells show brittle LEGO-brick failure, often along the numerous pit channels. In both species, cell walls (CWs) show distinct lamellar structures. These lamellae involve a helicoidal arrangement of cellulose macrofibrils as a recurring motif. Between the two nutshell species, these lamellae show differences in thickness and pitch angle, which can explain the different mechanical properties on the nanolevel. Our in-depth study of the two nutshell tissues highlights the role of cell form and their interlocking as well as plant CW composition and structure for mechanical protection. Understanding these plant shell concepts might inspire biomimetic material developments as well as using walnut and pistachio shell waste as sustainable raw material in future applications.

A belt for the cell: cellulosic wall thickenings and their role in morphogenesis of the 3D puzzle cells in walnut shells.

Walnut (Juglans regia) kernels are protected by a tough shell consisting of polylobate sclereids that interlock into a 3D puzzle. The shape transformations from isodiametric to lobed cells is well documented for 2D pavement cells, but not for 3D puzzle sclereids. Here, we study the morphogenesis of these cells by using a combination of different imaging techniques. Serial face-microtomy enabled us to reconstruct tissue growth of whole walnut fruits in 3D, and serial block face-scanning electron microscopy exposed cell shapes and their transformation in 3D during shell tissue development. In combination with Raman and fluorescence microscopy, we revealed multiple loops of cellulosic thickenings in cell walls, acting as stiff restrictions during cell growth and leading to the lobed cell shape. Our findings contribute to a better understanding of the 3D shape transformation of polylobate sclereids and the role of pectin and cellulose within this process.

Topological Interlocking and Geometric Stiffening as Complementary Strategies for Strong Plant Shells.

Many organisms encapsulate their embryos in hard, protective shells. While birds and reptiles largely rely on mineralized shells, plants often develop highly robust lignocellulosic shells. Despite the abundance of hard plant shells, particularly nutshells, it remains unclear which fundamental properties drive their mechanical stability. This multiscale analysis of six prominent (nut)shells (pine, pistachio, walnut, pecan, hazelnut, and macadamia) reveals geometric and structural strengthening mechanisms on the cellular and macroscopic length scales. The strongest tissues, found in walnut and pistachio, exploit the topological interlocking of 3D-puzzle cells and thereby outperform the fiber-reinforced structure of macadamia under tensile and compressive loading. On the macroscopic scale, strengthening occurs via an increased shell thickness, spherical shape, small size, and a lack of extended sutures. These functional interrelations suggest that simple geometric modifications are a powerful and resource-efficient strategy for plants to enhance the fracture resistance of entire shells and their tissues. Understanding the interplay between structure, geometry, and mechanics in hard plant shells provides new perspectives on the evolutionary diversification of hard seed coats, as well as insights for nutshell-based material applications.

From the Soft to the Hard: Changes in Microchemistry During Cell Wall Maturation of Walnut Shells.

The walnut shell is a hard and protective layer that provides an essential barrier between the seed and its environment. The shell is based on only one unit cell type: the polylobate sclerenchyma cell. For a better understanding of the interlocked walnut shell tissue, we investigate the structural and compositional changes during the development of the shell from the soft to the hard state. Structural changes at the macro level are explored by X-ray tomography and on the cell and cell wall level various microscopic techniques are applied. Walnut shell development takes place beneath the outer green husk, which protects and delivers components during the development of the walnut. The cells toward this outer green husk have the thickest and most lignified cell walls. With maturation secondary cell wall thickening takes place and the amount of all cell wall components (cellulose, hemicelluloses and especially lignin) is increased as revealed by FTIR microscopy. Focusing on the cell wall level, Raman imaging showed that lignin is deposited first into the pectin network between the cells and cell corners, at the very beginning of secondary cell wall formation. Furthermore, Raman imaging of fluorescence visualized numerous pits as a network of channels, connecting all the interlocked polylobate walnut shells. In the final mature stage, fluorescence increased throughout the cell wall and a fluorescent layer was detected toward the lumen in the inner part. This accumulation of aromatic components is reminiscent of heartwood formation of trees and is suggested to improve protection properties of the mature walnut shell. Understanding the walnut shell and its development will inspire biomimetic material design and packaging concepts, but is also important for waste valorization, considering that walnuts are the most widespread tree nuts in the world.

The Puzzle of the Walnut Shell: A Novel Cell Type with Interlocked Packing.

The outer protective shells of nuts can have remarkable toughness and strength, which are typically achieved by a layered arrangement of sclerenchyma cells and fibers with a polygonal form. Here, the tissue structure of walnut shells is analyzed in depth, revealing that the shells consist of a single, never reported cell type: the polylobate sclereid cells. These irregularly lobed cells with concave and convex parts are on average interlocked with 14 neighboring cells. The result is an intricate arrangement that cannot be disassembled when conceived as a 3D puzzle. Mechanical testing reveals a significantly higher ultimate tensile strength of the interlocked walnut cell tissue compared to the sclerenchyma tissue of a pine seed coat lacking the lobed cell structure. The higher strength value of the walnut shell is explained by the observation that the crack cannot simply detach intact cells but has to cut through the lobes due to the interlocking. Understanding the identified nutshell structure and its development will inspire biomimetic material design and packaging concepts. Furthermore, these unique unit cells might be of special interest for utilizing nutshells in terms of food waste valorization, considering that walnuts are the most widespread tree nuts in the world.

Agitation Modules: Flexible Means to Accelerate Automated Freeze Substitution.

For ultrafast fixation of biological samples to avoid artifacts, high-pressure freezing (HPF) followed by freeze substitution (FS) is preferred over chemical fixation at room temperature. After HPF, samples are maintained at low temperature during dehydration and fixation, while avoiding damaging recrystallization. This is a notoriously slow process. McDonald and Webb demonstrated, in 2011, that sample agitation during FS dramatically reduces the necessary time. Then, in 2015, we (H.G. and S.R.) introduced an agitation module into the cryochamber of an automated FS unit and demonstrated that the preparation of algae could be shortened from days to a couple of hours. We argued that variability in the processing, reproducibility, and safety issues are better addressed using automated FS units. For dissemination, we started low-cost manufacturing of agitation modules for two of the most widely used FS units, the Automatic Freeze Substitution Systems, AFS(1) and AFS2, from Leica Microsystems, using three dimensional (3D)-printing of the major components. To test them, several labs independently used the modules on a wide variety of specimens that had previously been processed by manual agitation, or without agitation. We demonstrate that automated processing with sample agitation saves time, increases flexibility with respect to sample requirements and protocols, and produces data of at least as good quality as other approaches.

Lichen secondary metabolites affect growth of Physcomitrella patens by allelopathy.

Lichen secondary metabolites can function as allelochemicals and affect the development and growth of neighboring bryophytes, fungi, vascular plants, microorganisms, and even other lichens. Lichen overgrowth on bryophytes is frequently observed in nature even though mosses grow faster than lichens, but there is still little information on the interactions between lichens and bryophytes.In the present study, we used extracts from six lichen thalli containing secondary metabolites like usnic acid, protocetraric acid, atranorin, lecanoric acid, nortistic acid, and thamnolic acid. To observe the influence of these metabolites on bryophytes, the moss Physcomitrella patens was cultivated for 5 weeks under laboratory conditions and treated with lichen extracts. Toxicity of natural mixtures of secondary metabolites was tested at three selected doses (0.001, 0.01, and 0.1 %). When the mixture contained substantial amounts of usnic acid, we observed growth inhibition of protonemata and reduced development of gametophores. Significant differences in cell lengths and widths were also noticed. Furthermore, usnic acid had a strong effect on cell division in protonemata suggesting a strong impact on the early stages of bryophyte development by allelochemicals contained in the lichen secondary metabolites.Biological activities of lichen secondary metabolites were confirmed in several studies such as antiviral, antibacterial, antitumor, antiherbivore, antioxidant, antipyretic, and analgetic action or photoprotection. This work aimed to expand the knowledge on allelopathic effects on bryophyte growth.

Limited accumulation of copper in heavy metal adapted mosses.

Copper is an essential micronutrient but has toxic effects at high concentrations. Bryophytes are remarkably tolerant to elevated levels of copper but we wondered if this tolerance might be species dependent. Therefore, in three moss species, Physcomitrella patens, Mielichhoferia elongata and Pohlia drummondii, the accumulation of copper was compared with semiquantitative SEM-EDX analyses after six weeks of cultivation on copper containing media. We investigated the role of the copper-linked anion and applied copper as CuCl2, CuSO4 and CuEDTA, respectively. Line scans along the growth axis of moss gametophores allowed for a detailed analysis of copper detection from the base towards the tip. Mosses originating from metal-containing habitats (i.e. M. elongata and P. drummondii) revealed a lower accumulation of copper when compared to the non-adapted P. patens. CuEDTA had a shielding effect in all three species and copper levels differed greatly from CuCl2 or CuSO4. The detection of reactive oxygen species (ROS), H2O2 and O2(-), was further used to indicate stress levels in the gametophore stems. ROS staining was increased along the whole stem and the tip in the non-adapted species P. patens whereas the tolerant species M. elongata and P. drummondii generally showed less staining located mainly at the base of the stem. We discuss the relation between metal accumulation and ROS production using indicator dyes in the three moss species. As moss gametophores are very delicate structures, ROS staining provide an excellent alternative to spectrophotometric analyses to estimate stress levels.