X-ray microscopy enables multiscale high-resolution 3D imaging of plant cells, tissues, and organs.

Capturing complete internal anatomies of plant organs and tissues within their relevant morphological context remains a key challenge in plant science. While plant growth and development are inherently multiscale, conventional light, fluorescence, and electron microscopy platforms are typically limited to imaging of plant microstructure from small flat samples that lack a direct spatial context to, and represent only a small portion of, the relevant plant macrostructures. We demonstrate technical advances with a lab-based X-ray microscope (XRM) that bridge the imaging gap by providing multiscale high-resolution three-dimensional (3D) volumes of intact plant samples from the cell to the whole plant level. Serial imaging of a single sample is shown to provide sub-micron 3D volumes co-registered with lower magnification scans for explicit contextual reference. High-quality 3D volume data from our enhanced methods facilitate sophisticated and effective computational segmentation. Advances in sample preparation make multimodal correlative imaging workflows possible, where a single resin-embedded plant sample is scanned via XRM to generate a 3D cell-level map, and then used to identify and zoom in on sub-cellular regions of interest for high-resolution scanning electron microscopy. In total, we present the methodologies for use of XRM in the multiscale and multimodal analysis of 3D plant features using numerous economically and scientifically important plant systems.

Plant mitochondria - past, present and future.

The study of plant mitochondria started in earnest around 1950 with the first isolations of mitochondria from animal and plant tissues. The first 35 years were spent establishing the basic properties of plant mitochondria and plant respiration using biochemical and physiological approaches. A number of unique properties (compared to mammalian mitochondria) were observed: (i) the ability to oxidize malate, glycine and cytosolic NAD(P)H at high rates; (ii) the partial insensitivity to rotenone, which turned out to be due to the presence of a second NADH dehydrogenase on the inner surface of the inner mitochondrial membrane in addition to the classical Complex I NADH dehydrogenase; and (iii) the partial insensitivity to cyanide, which turned out to be due to an alternative oxidase, which is also located on the inner surface of the inner mitochondrial membrane, in addition to the classical Complex IV, cytochrome oxidase. With the appearance of molecular biology methods around 1985, followed by genomics, further unique properties were discovered: (iv) plant mitochondrial DNA (mtDNA) is 10-600 times larger than the mammalian mtDNA, yet it only contains approximately 50% more genes; (v) plant mtDNA has kept the standard genetic code, and it has a low divergence rate with respect to point mutations, but a high recombinatorial activity; (vi) mitochondrial mRNA maturation includes a uniquely complex set of activities for processing, splicing and editing (at hundreds of sites); (vii) recombination in mtDNA creates novel reading frames that can produce male sterility; and (viii) plant mitochondria have a large proteome with 2000-3000 different proteins containing many unique proteins such as 200-300 pentatricopeptide repeat proteins. We describe the present and fairly detailed picture of the structure and function of plant mitochondria and how the unique properties make their metabolism more flexible allowing them to be involved in many diverse processes in the plant cell, such as photosynthesis, photorespiration, CAM and C4 metabolism, heat production, temperature control, stress resistance mechanisms, programmed cell death and genomic evolution. However, it is still a challenge to understand how the regulation of metabolism and mtDNA expression works at the cellular level and how retrograde signaling from the mitochondria coordinates all those processes.

Spotting plants' microfilament morphologies and nanostructures.

The tracheary system of plant leaves is composed of a cellulose skeleton with diverse hierarchical structures. It is built of polygonally bent helical microfilaments of cellulose-based nanostructures coated by different layers, which provide them high compression resistance, elasticity, and roughness. Their function includes the transport of water and nutrients from the roots to the leaves. Unveiling details about local interactions of tracheary elements with surrounding material, which varies between plants due to adaptation to different environments, is crucial for understanding ascending fluid transport and for tracheary mechanical strength relevant to potential applications. Here we show that plant tracheary microfilaments, collected from Agapanthus africanus and Ornithogalum thyrsoides leaves, have different surface morphologies, revealed by nematic liquid crystal droplets. This results in diverse interactions among microfilaments and with the environment; the differences translate to diverse mechanical properties of entangled microfilaments and their potential applications. The presented study also introduces routes for accurate characterization of plants' microfilaments.

The structural and functional domains of plant thylakoid membranes.

In plants, the stacking of part of the photosynthetic thylakoid membrane generates two main subcompartments: the stacked grana core and unstacked stroma lamellae. However, a third distinct domain, the grana margin, has been postulated but its structural and functional identity remains elusive. Here, an optimized thylakoid fragmentation procedure combined with detailed ultrastructural, biochemical, and functional analyses reveals the distinct composition of grana margins. It is enriched with lipids, cytochrome b6 f complex, and ATPase while depleted in photosystems and light-harvesting complexes. A quantitative method is introduced that is based on Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) and dot immunoblotting for quantifying various photosystem II (PSII) assembly forms in different thylakoid subcompartments. The results indicate that the grana margin functions as a degradation and disassembly zone for photodamaged PSII. In contrast, the stacked grana core region contains fully assembled and functional PSII holocomplexes. The stroma lamellae, finally, contain monomeric PSII as well as a significant fraction of dimeric holocomplexes that identify this membrane area as the PSII repair zone. This structural organization and the heterogeneous PSII distribution support the idea that the stacking of thylakoid membranes leads to a division of labor that establishes distinct membrane areas with specific functions.

Visualising endocytosis in plants: past, present, and future.

Chris Hawes had a lively fascination for the immensely complex organisation of the endomembrane system, including the process of endocytosis. This is the method by which eukaryotic cells internalise membrane proteins, lipids, carbohydrates, and cell wall enzymes from the cell surface through membrane bound vesicles. Endocytosis occurs progressively, starting with early membrane deformation, scission, and finally the release of the vesicle into the cytoplasm. Next to secretion, endocytosis allows the cell to control the proteome composition of its inner and outer surface membrane and as such, its communication with the outside world. Whereas endocytosis was initially considered theoretically impossible in plants due to their high turgor pressure, it is now established as essential for plant life. Furthermore, endocytosis remains a highly active field of research, both in yeast, animal, and plant model systems. Over the past three decades, the tools and techniques used to visualise, quantify, and characterise endocytosis have resulted in an increasingly higher spatiotemporal understanding of this process. Here we provide a brief history of plant endocytosis research from the time when Chris Hawes was investigating the process, to the current state-of-the-art in the field. We will end this chapter with a discussion on some promising future developments for plant endocytosis research. LAY DESCRIPTION: Endocytosis is a key process whereby eukaryotic cells can selectively take up membrane proteins, extracellular material and lipids. As this process controls the abundance and protein composition of the plasma membrane, it also controls the communication of the cell with the outside world. Whereas endocytosis was initially considered theoretically impossible in plants due to their high turgor pressure, it is now established as essential for plant life. Today, endocytosis remains a highly active field of research, both in yeast, animal, and plant model systems. Endocytosis was one of the favourite research topics of Chris Hawes, which is why this mini-review is part of the Festschrift issue in his honour. We provide here a brief history of plant endocytosis research from the time when Chris Hawes was investigating the process, to the current state-of-the-art in the field. Over the past three decades, the tools and techniques that were developed to visualise, quantify, and characterise endocytosis have allowed to achieve an increasingly higher spatiotemporal understanding of this process. We end this chapter with a discussion on some promising future developments for plant endocytosis research.

A journey through a plant cytoskeleton: Hot spots in signaling and functioning.

This survey paper contains a brief analysis of publications included in the special issue of the scientific journal Cell Biology International titled "Plant Cytoskeleton Structure, Dynamics and Functions". The manuscripts in this special issue reflect some new aspects of plant cytoskeleton organization, signaling and functioning, and results from different Ukrainian research groups, and focuses on bringing together scientists working across different instrumental scales.

Xyloglucan in the primary cell wall: assessment by FESEM, selective enzyme digestions and nanogold affinity tags.

Xyloglucan has been hypothesized to bind extensively to cellulose microfibril surfaces and to tether microfibrils into a load-bearing network, thereby playing a central role in wall mechanics and growth, but this view is challenged by newer results. Here we combined high-resolution imaging by field emission scanning electron microscopy (FESEM) with nanogold affinity tags and selective endoglucanase treatments to assess the spatial location and conformation of xyloglucan in onion cell walls. FESEM imaging of xyloglucanase-digested cell walls revealed an altered microfibril organization but did not yield clear evidence of xyloglucan conformations. Backscattered electron detection provided excellent detection of nanogold affinity tags in the context of wall fibrillar organization. Labelling with xyloglucan-specific CBM76 conjugated with nanogold showed that xyloglucans were associated with fibril surfaces in both extended and coiled conformations, but tethered configurations were not observed. Labelling with nanogold-conjugated CBM3, which binds the hydrophobic surface of crystalline cellulose, was infrequent until the wall was predigested with xyloglucanase, whereupon microfibril labelling was extensive. When tamarind xyloglucan was allowed to bind to xyloglucan-depleted onion walls, CBM76 labelling gave positive evidence for xyloglucans in both extended and coiled conformations, yet xyloglucan chains were not directly visible by FESEM. These results indicate that an appreciable, but still small, surface of cellulose microfibrils in the onion wall is tightly bound with extended xyloglucan chains and that some of the xyloglucan has a coiled conformation.

Vacuolar membrane structures and their roles in plant-pathogen interactions.

KEY MESSAGE: Short review focussing on the role and targeting of vacuolar substructure in plant immunity and pathogenesis. Plants lack specialized immune cells, therefore each plant cell must defend itself against invading pathogens. A typical plant defense strategy is the hypersensitive response that results in host cell death at the site of infection, a process largely regulated by the vacuole. In plant cells, the vacuole is a vital organelle that plays a central role in numerous fundamental processes, such as development, reproduction, and cellular responses to biotic and abiotic stimuli. It shows divergent membranous structures that are continuously transforming. Recent technical advances in visualization and live-cell imaging have significantly altered our view of the vacuolar structures and their dynamics. Understanding the active nature of the vacuolar structures and the mechanisms of vacuole-mediated defense responses is of great importance in understanding plant-pathogen interactions. In this review, we present an overview of the current knowledge about the vacuole and its internal structures, as well as their role in plant-microbe interactions. There is so far limited information on the modulation of the vacuolar structures by pathogens, but recent research has identified the vacuole as a possible target of microbial interference.

Chloroplast ultrastructure in plants.

The chloroplast organelle in mesophyll cells of higher plants represents a sunlight-driven metabolic factory that eventually fuels life on our planet. Knowledge of the ultrastructure and the dynamics of this unique organelle is essential to understanding its function in an ever-changing and challenging environment. Recent technological developments promise unprecedented insights into chloroplast architecture and its functionality. The review highlights these new methodical approaches and provides structural models based on recent findings about the plasticity of the thylakoid membrane system in response to different light regimes. Furthermore, the potential role of the lipid droplets plastoglobuli is discussed. It is emphasized that detailed structural insights are necessary on different levels ranging from molecules to entire membrane systems for a holistic understanding of chloroplast function.

Synchrotron-Based X-Ray Fluorescence Microscopy as a Technique for Imaging of Elements in Plants.

Understanding the distribution of elements within plant tissues is important across a range of fields in plant science. In this review, we examine synchrotron-based x-ray fluorescence microscopy (XFM) as an elemental imaging technique in plant sciences, considering both its historical and current uses as well as discussing emerging approaches. XFM offers several unique capabilities of interest to plant scientists, including in vivo analyses at room temperature and pressure, good detection limits (approximately 1-100 mg kg-1), and excellent resolution (down to 50 nm). This has permitted its use in a range of studies, including for functional characterization in molecular biology, examining the distribution of nutrients in food products, understanding the movement of foliar fertilizers, investigating the behavior of engineered nanoparticles, elucidating the toxic effects of metal(loid)s in agronomic plant species, and studying the unique properties of hyperaccumulating plants. We anticipate that continuing technological advances at XFM beamlines also will provide new opportunities moving into the future, such as for high-throughput screening in molecular biology, the use of exotic metal tags for protein localization, and enabling time-resolved, in vivo analyses of living plants. By examining current and potential future applications, we hope to encourage further XFM studies in plant sciences by highlighting the versatility of this approach.

Plant Lipid Droplets and Their Associated Proteins: Potential for Rapid Advances.

Cytoplasmic lipid droplets (LDs) of neutral lipids (triacylglycerols [TAGs], sterylesters, etc.) are reserves of high-energy metabolites and other constituents for future needs. They are present in diverse cells of eukaryotes and prokaryotes. An LD has a core of neutral lipids enclosed with a monolayer of phospholipids and proteins, which play structural and/or metabolic roles. During the past 3 decades, studies of LDs in diverse organisms have blossomed after they were found to be involved in prevalent human diseases and industrial uses. LDs in plant seeds were studied before those in mammals and microbes, and the latter studies have since moved forward. Plant LDs carry a hallmark protein called oleosin, which has a long hydrophobic hairpin penetrating the TAG core and stabilizing the LD. The oleosin gene first appeared in green algae and has evolved in enhancing promoter strength, tandem repeats, and/or expression specificity, leading to the appearance of new LD organelles, such as tapetosomes in Brassicaceae. The synthesis of LDs occurs with TAG-synthesizing enzymes on the endoplasmic reticulum (ER), and nascent TAGs are sequestered in the acyl moiety region between the bilayers of phospholipids, which results in ER-LD swelling. Oleosin is synthesized on the cytosol side of the ER and extracts the LD from the ER-LD to cytosol. This extraction of LD to the cytosol is controlled solely by the innate properties of oleosin, and modified oleosin can redirect the LD to the ER lumen and then vacuoles. The breakdown of LDs requires lipase associating with core retromer and binding to peroxisomes, which then send the enzyme to LDs via tubular extensions. Two groups of LD-associated proteins, caleosin/dioxygenase/steroleosin and LD/oil body-associated proteins, participate in cellular stress defenses via enzymic activities and binding, respectively. The surface of LDs in all plant cells may be an inert refuge for these and other proteins, which exert functions on diverse cell components. Oleosin-LDs have been explored for commercial applications; successes in their uses will rely on overcoming conceptual and technical difficulties.

Morphology of the megaspore Lagenoisporites magnus (Chi and Hills 1976) Candilier et al. (1982), from the Carboniferous (lower Mississippian: mid-upper Tournaisian) of Bolivia.

The morphology and structure of megaspores assigned to Lagenoisporites magnus from the Toregua Formation, Retama Group, mid-upper Tournaisian of Bolivia were studied. The analysis was performed with light, fluorescence and scanning electron microscopy. Megaspores were laterally compressed and presented a spherical body with a proximal gula, of the hologula type. Gula had verrucae ornamentation and the spore body presented complex processes consisting of a bulbous base and an internally partitioned projection with sharp apex. In addition to this main ornamentation, perforations were present throughout the spore surface. Megaspores showed well marked curvaturae perfectae due to the abrupt transition existing between the gula ornamentation and the spore body processes. These megaspores were assigned to heterosporous arborescent lycopsids of the Lepidocarpaceae family, as in section view, exospore structure presented a three-dimensional network of fused elements. Likewise, due to a similarity found between sporoderm and Isoetes L. structure, it is evident that megaspores structure has remained intact inside the heterosporous lycopsids. Therefore; the L. magnus structure not only would confirm its affinity with the Lycophyta fossils but also with the living ones.

Characterization and Phytotoxicity Assessment of Essential Oils from Plant Byproducts.

The present work describes the chemical characterization and the phytotoxicity assessment of essential oils (EOs) obtained from spent materials or pruning waste of four plant species: Zingiber officinale Roscoe used in the juicing industry, Pistacia vera L. var. Bronte used in the food industry, discarded material of industrial hemp (Cannabis sativa L. var. Futura 75), and pruning waste from Cupressus sempervirens L. The phytochemical profile of the EOs was evaluated by gas chromatographic flame ionization detection (GC-FID) and GC-MS analyses, which highlighted the presence of several compounds with a wide range of biological activities. Among them, application possibilities in agriculture were evaluated by studying the phytotoxic activity in vitro against germination and initial radical growth of several seeds such as Raphanus sativus L., Lepidium sativum L., Lactuca sativa L., Solanum lycopersicum L., Lolium multiflorum Lam., and Portulaca oleracea L.

Autophagy in plant pathogenic fungi.

Autophagy is a conserved cellular process that degrades cytoplasmic constituents in vacuoles. Plant pathogenic fungi develop special infection structures and/or secrete a range of enzymes to invade their plant hosts. It has been demonstrated that monitoring autophagy processes can be extremely useful in visualizing the sequence of events leading to pathogenicity of plant pathogenic fungi. In this review, we introduce the molecular mechanisms involved in autophagy. In addition, we explore the relationship between autophagy and pathogenicity in plant pathogenic fungi. Finally, we discuss the various experimental strategies available for use in the study of autophagy in plant pathogenic fungi.

Chloroplast DNA Dynamics: Copy Number, Quality Control and Degradation.

Endosymbiotically originated chloroplast DNA (cpDNA) encodes part of the genetic information needed to fulfill chloroplast function, including fundamental processes such as photosynthesis. In the last two decades, advances in genome analysis led to the identification of a considerable number of cpDNA sequences from various species. While these data provided the consensus features of cpDNA organization and chloroplast evolution in plants, how cpDNA is maintained through development and is inherited remains to be fully understood. In particular, the fact that cpDNA exists as multiple copies despite its limited genetic capacity raises the important question of how copy number is maintained or whether cpDNA is subjected to quantitative fluctuation or even developmental degradation. For example, cpDNA is abundant in leaves, where it forms punctate structures called nucleoids, which seemingly alter their morphologies and numbers depending on the developmental status of the chloroplast. In this review, we summarize our current understanding of 'cpDNA dynamics', focusing on the changes in DNA abundance. A special focus is given to the cpDNA degradation mechanism, which appears to be mediated by Defective in Pollen organelle DNA degradation 1 (DPD1), a recently discovered organelle exonuclease. The physiological significance of cpDNA degradation in flowering plants is also discussed.

Natural Plant Materials as Dielectric Layer for Highly Sensitive Flexible Electronic Skin.

Nature has long offered human beings with useful materials. Herein, plant materials including flowers and leaves have been directly used as the dielectric material in flexible capacitive electronic skin (e-skin), which simply consists of a dried flower petal or leaf sandwiched by two flexible electrodes. The plant material is a 3D cell wall network which plays like a compressible metamaterial that elastically collapses upon pressing plus some specific surface structures, and thus the device can sensitively respond to pressure. The device works over a broad-pressure range from 0.6 Pa to 115 kPa with a maximum sensitivity of 1.54 kPa-1 , and shows high stability over 5000 cyclic pressings or bends. The natural-material-based e-skin has been applied in touch sensing, motion monitoring, gas flow detection, and the spatial distribution of pressure. As the foam-like structure is ubiquitous in plants, a general strategy for a green, cost-effective, and scalable approach to make flexible e-skins is offered here.

The future has roots in the past: the ideas and scientists that shaped mycorrhizal research.

Contents Summary 982 I. Introduction 982 II. The portraits of our ancestors: a gallery of ideas from more than 100 years of mycorrhizal research 983 III. Mycorrhizal fungi in the 'omics' era: first puzzle, how to name mycorrhizal fungi 985 IV. Signalling: a central question of our time? 987 V. The colonization process: how cellular studies predicted future 'omics' data 989 VI. The genetics underlying colonization events 991 VII. Concluding thoughts: chance and needs in mycorrhizal symbioses 992 Acknowledgements 992 References 992 SUMMARY: Our knowledge of mycorrhizas dates back to at least 150 years ago, when the plant pathologists A. B. Frank and G. Gibelli described the surprisingly morphology of forest tree roots surrounded by a fungal mantle. Compared with this history, our molecular study of mycorrhizas remains a young science. To trace the history of mycorrhizal research, from its roots in the distant past, to the present and the future, this review outlines a few topics that were already central in the 19th century and were seminal in revealing the biological meaning of mycorrhizal associations. These include investigations of nutrient exchange between partners, plant responses to mycorrhizal fungi, and the identity and evolution of mycorrhizal symbionts as just a few examples of how the most recent molecular studies of mycorrhizal biology sprouted from the roots of past research. In addition to clarifying the ecological role of mycorrhizas, some of the recent results have changed the perception of the relevance of mycorrhizas in the scientific community, and in the whole of society. Looking to past knowledge while foreseeing strategies for the next steps can help us catch a glimpse of the future of mycorrhizal research.

Unity in diversity: structural and functional insights into the ancient partnerships between plants and fungi.

Contents Summary 996 I. Introduction 996 II. An ancient, and diverse, symbiosis 998 III. Structural diversity in ancient plant-fungal partnerships 1000 IV. Mycorrhizal unity in host plant nutrition 1002 V. Plant-to-fungus carbon transfer 1003 VI. From individuals to networks 1003 VII. Diverse responses of mycorrhizal functioning to dynamic environments 1006 VIII. Summary of future research direction 1007 Acknowledgements 1006 References 1006 SUMMARY: Mycorrhizal symbiosis is an ancient and widespread mutualism between plants and fungi that facilitated plant terrestrialisation > 500 million years ago, with key roles in ecosystem functioning at multiple scales. Central to the symbiosis is the bidirectional exchange of plant-fixed carbon for fungal-acquired nutrients. Within this unifying role of mycorrhizas, considerable diversity in structure and function reflects the diversity of the partners involved. Early diverging plants form mutualisms not only with arbuscular mycorrhizal Glomeromycotina fungi, but also with poorly characterised Mucoromycotina, which may also colonise the roots of 'higher' plants as fine root endophytes. Functional diversity in these symbioses depends on both fungal and plant life histories and is influenced by the environment. Recent studies have highlighted the roles of lipids/fatty acids in plant-to-fungus carbon transport and potential contributions of Glomeromycotina fungi to plant nitrogen nutrition. Together with emerging appreciation of mycorrhizal networks as multi-species resource-sharing systems, these insights are broadening our views on mycorrhizas and their roles in nutrient cycling. It is crucial that the diverse array of biotic and abiotic factors that together shape the dynamics of carbon-for-nutrient exchange between plants and fungi are integrated, in addition to embracing the unfolding and potentially key role of Mucoromycotina fungi in these processes.

Characterization of a newly isolated freshwater Eustigmatophyte alga capable of utilizing far-red light as its sole light source.

Oxygenic phototrophs typically utilize visible light (400-700 nm) to drive photosynthesis. However, a large fraction of the energy in sunlight is contained in the far-red region, which encompasses light beyond 700 nm. In nature, certain niche environments contain high levels of this far-red light due to filtering by other phototrophs, and in these environments, organisms with photosynthetic antenna systems adapted to absorbing far-red light are able to thrive. We used selective far-red light conditions to isolate such organisms in environmental samples. One cultured organism, the Eustigmatophyte alga Forest Park Isolate 5 (FP5), is able to absorb far-red light using a chlorophyll (Chl) a-containing antenna complex, and is able to grow under solely far-red light. Here we characterize the antenna system from this organism, which is able to shift the absorption of Chl a to >705 nm.

Evolution of root apical meristem structures in vascular plants: plasmodesmatal networks.

PREMISE OF THE STUDY: The apical meristem generates indeterminate apical growth of the stem and root of vascular plants. Our previous examination showed that shoot apical meristems (SAMs) can be classified into two types based on plasmodesmatal networks (PNs), which are important elements in symplasmic signaling pathways within the apical meristem. Here, we examined the PNs of root apical meristems (RAMs) in comparison with those of SAMs. METHODS: Root apical meristems of 18 families and 22 species of lycophytes and euphyllophytes were analyzed. Plasmodesmata (PD) in cell walls in median longitudinal sections of RAMs were enumerated using transmission electron micrographs, and the PD density per 1 µm2 of each cell wall was calculated. KEY RESULTS: Root apical meristems with prominent apical cells of monilophytes (euphyllophytes) and Selaginellaceae (lycophytes) had high PD densities, while RAMs with plural initial cells of gymnosperms and angiosperms (euphyllophytes), and of Lycopodiaceae and Isoetaceae (lycophytes) had low PD densities. This correlation between structures of apical meristems and PD densities is identical to that in SAMs already described. CONCLUSIONS: Irrespective of their diversified structures, the RAMs of vascular plants can be classified into two types with respect to PNs: the fern (monilophyte) type, which has a lineage-specific PN with only primary PD, and the seed-plant type, which has an interspecific PN with secondary PD in addition to primary PD. PNs may have played a key role in the evolution of apical meristems in vascular plants.