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.

Maintaining the structural and functional homeostasis of the plant endoplasmic reticulum.

The endoplasmic reticulum (ER) is a ubiquitous organelle that is vital to the life of eukaryotic cells. It synthesizes essential lipids and proteins and initiates the glycosylation of intracellular and surface proteins. As such, the ER is necessary for cell growth and communication with the external environment. The ER is also a highly dynamic organelle, whose structure is continuously remodeled through an interaction with the cytoskeleton and the action of specialized ER shapers. Recent and significant advances in ER studies have brought to light conserved and unique features underlying the structure and function of this organelle in plant cells. In this review, exciting developments in the understanding of the mechanisms for plant ER structural and functional homeostasis, particularly those that underpin ER network architecture and ER degradation, are presented and discussed.

Insights into Plant Programmed Cell Death Induced by Heavy Metals-Discovering a Terra Incognita.

Programmed cell death (PCD) is a process that plays a fundamental role in plant development and responses to biotic and abiotic stresses. Knowledge of plant PCD mechanisms is still very scarce and is incomparable to the large number of studies on PCD mechanisms in animals. Quick and accurate assays, e.g., the TUNEL assay, comet assay, and analysis of caspase-like enzyme activity, enable the differentiation of PCD from necrosis. Two main types of plant PCD, developmental (dPCD) regulated by internal factors, and environmental (ePCD) induced by external stimuli, are distinguished based on the differences in the expression of the conserved PCD-inducing genes. Abiotic stress factors, including heavy metals, induce necrosis or ePCD. Heavy metals induce PCD by triggering oxidative stress via reactive oxygen species (ROS) overproduction. ROS that are mainly produced by mitochondria modulate phytotoxicity mechanisms induced by heavy metals. Complex crosstalk between ROS, hormones (ethylene), nitric oxide (NO), and calcium ions evokes PCD, with proteases with caspase-like activity executing PCD in plant cells exposed to heavy metals. This pathway leads to very similar cytological hallmarks of heavy metal induced PCD to PCD induced by other abiotic factors. The forms, hallmarks, mechanisms, and genetic regulation of plant ePCD induced by abiotic stress are reviewed here in detail, with an emphasis on plant cell culture as a suitable model for PCD studies. The similarities and differences between plant and animal PCD are also discussed.

Sphingolipids in plants: a guidebook on their function in membrane architecture, cellular processes, and environmental or developmental responses.

Sphingolipids are fundamental lipids involved in various cellular, developmental and stress-response processes. As such, they orchestrate not only vital molecular mechanisms of living cells but also act in diseases, thus qualifying as potential pharmaceutical targets. Sphingolipids are universal to eukaryotes and are also present in some prokaryotes. Some sphingolipid structures are conserved between animals, plants and fungi, whereas others are found only in plants and fungi. In plants, the structural diversity of sphingolipids, as well as their downstream effectors and molecular and cellular mechanisms of action, are of tremendous interest to both basic and applied researchers, as about half of all small molecules in clinical use originate from plants. Here, we review recent advances towards a better understanding of the biosynthesis of sphingolipids, the diversity in their structures as well as their functional roles in membrane architecture, cellular processes such as membrane trafficking and cell polarity, and cell responses to environmental or developmental signals.

Imaging dataset of fresh hydrous plants obtained by field-emission scanning electron microscopy conducted using a protective NanoSuit.

Although scanning electron microscopy (SEM) can generate high-resolution images of nanosized objects, it requires a high vacuum to do so, which precludes direct observations of living organisms and often produces unwanted structural changes. It has previously been reported that a simple surface modification gives rise to a nanoscale layer, termed the "NanoSuit", which can keep small animals alive under the high vacuum required for field-emission scanning electron microscopy (FE-SEM). We have previously applied this technique to plants, and successfully observed healthy petals in a fully hydrated state using SEM. The flower petals protected with the NanoSuit appeared intact, although we still lack a fundamental understanding of the images of other plants observed using FE-SEM. This report presents and evaluates a rich set of images, acquired using the NanoSuit, for a taxonomically diverse set of plant species. This dataset of images allows the surface features of various plants to be analyzed and thus provides a further complementary morphological profile. Image data can be accessed and viewed through Figshare (https://doi.org/10.6084/m9.figshare.c.4446026.v1).

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.

Response of submerged macrophytes and leaf biofilms to the decline phase of Microcystis aeruginosa: Antioxidant response, ultrastructure, microbial properties, and potential mechanism.

Decaying cyanobacterial blooms carry a potential risk for submerged macrophyte and periphyton biofilms in aquatic environments. This study comprehensively studied the responses in growth, oxidative response, detoxification pathway, and ultrastructure characteristics of aquatic plants to Microcystis aeruginosa (M. aeruginosa) exudates and extracts released during the decline phase. Particular emphasis was placed on the variation of extracellular polymeric substances (EPS) and quorum-sensing signaling molecules. The results showed that superoxide dismutase, peroxidase, and glutathione S-transferase were significantly induced as antioxidant response, and the malondialdehyde content increased. Increased content of MC-LR (1.129 µg L-1) and NH4+-N (1.35 mg L-1) were found in the decline phase of M. aeruginosa, which played a vital role in the damage to submerged plants. In addition, a change in the amount of osmiophilic granules and a variation of organelles and membranes was observed. A broad distribution of α-d-glucopyranose polysaccharides was dominant and aggregated into clusters in biofilm EPS in response to exposure to decaying M. aeruginosa. Furthermore, exposure to exudates and extracts changed the abundance and structure of the microbial biofilm community. Increased contents of N-acylated-L-homoserine lactone signal molecule might result in a variation of biofilm EPS production in response to decaying M. aeruginosa. These results expand the understanding of how submerged macrophyte and periphyton biofilms respond to environmental stress caused by exudates and extracts of decaying M. aeruginosa.

Real-time and noninvasive detection of UV-Induced deep tissue damage using electrical tattoos.

Understanding longterm deep tissue damage caused by UV radiation is imperative for ensuring the health and safety of living organisms that are regularly exposed to radiation sources. While existing UV dosimeters can quantify the cumulative amount of radiation to which an organism is exposed, these sensors cannot reveal the presence and extent of internal tissue damage caused by such exposure. Here we describe a method that uses conducting polymer tattoos to detect UV radiation-induced deep tissue damage in living organisms using bioimpedance analysis (BIA), which allows for noninvasive, real-time measurements of body composition and point-of-care assessment of clinical condition. To establish a performance baseline for this method, we quantify the effects of UVA radiation on live plant leaves. Low-energy UVA waves penetrate further into biological tissue, as compared to UVB, UVC and ionizing radiation, and cause longlasting deep tissue damage that cannot be immediately and readily detected using surface-sensitive techniques, such as photogrammetry and epidermal sensors. We show that single-frequency bioimpedance analysis allows for sensitive, real-time monitoring of UVA damage: as UVA dose increases, the bioimpedance of a plant leaf measured at a frequency of 1 kHz linearly decreases until the extent of radiation damage saturates and the specimen is effectively necrotized. We establish a strong correlation between radiation fluence, internal biological damage and the bioimpedance signal measured using our conducting polymer tattoos, which supports the efficacy of our method as a new type of internal biodosimetry.

Structural Biology and Electron Microscopy of the Autophagy Molecular Machinery.

Autophagy is a highly regulated bulk degradation process that plays a key role in the maintenance of cellular homeostasis. During autophagy, a double membrane-bound compartment termed the autophagosome is formed through de novo nucleation and assembly of membrane sources to engulf unwanted cytoplasmic components and targets them to the lysosome or vacuole for degradation. Central to this process are the autophagy-related (ATG) proteins, which play a critical role in plant fitness, immunity, and environmental stress response. Over the past few years, cryo-electron microscopy (cryo-EM) and single-particle analysis has matured into a powerful and versatile technique for the structural determination of protein complexes at high resolution and has contributed greatly to our current understanding of the molecular mechanisms underlying autophagosome biogenesis. Here we describe the plant-specific ATG proteins and summarize recent structural and mechanistic studies on the protein machinery involved in autophagy initiation with an emphasis on those by single-particle analysis.

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.

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.

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.

Essential Methods of Plant Sample Preparation for Light Microscopy.

There are various preparatory techniques for light microscopy permitting access to the inner structure of plant body and its development. Minute objects might be processed as whole-mount preparations, while voluminous ones should be separated into smaller pieces. Here we summarize some of the "classical" techniques to cut more voluminous objects into slices and access their inner structure either for simple anatomical analysis or for further processing (e.g., histochemistry, immunohistochemistry, in situ hybridization, enzyme histochemistry).

Selected Simple Methods of Plant Cell Wall Histochemistry and Staining for Light Microscopy.

Histochemical methods allow for identification and localization of various components within the tissue. Such information on the spatial heterogeneity is not available with biochemical methods. However, there is limitation of the specificity of such detection in context of complex tissue, which is important to consider, and interpretations of the results should regard suitable control treatments if possible. Such methods are valuable extension to specific optical and spectroscopic analytical methods. Here we present a set of selected simple methods of staining and histochemical tests with comments based on our laboratory experience.

Chemical Fixation, Immunofluorescence, and Immunogold Labeling of Electron Microscopical Sections.

Knowledge about the spatiotemporal distribution patterns of proteins and other molecules of the cell is essential for understanding their function. A widely used technique is immunolabeling which uses specific antibodies to reveal the distribution of molecular components at various structural levels. Immunofluorescence gives an overview about the distribution of molecules at the level of the fluorescence or confocal laser scanning microscope. Electron microscopy offers the highest resolution of morphological techniques and is thus an indispensable tool for the analysis of molecule distribution patterns at the subcellular level. In this chapter we describe selected routine methods for immunofluorescence and for labeling ultrathin sections of resin-embedded material with antibodies conjugated to colloidal gold, including protocols for chemical fixation, embedding, and sectioning.

Essential Methods of Plant Sample Preparation for High-Resolution Scanning Electron Microscopy at Room Temperature.

In this chapter, conventional techniques are described for the preparation of plant samples at room temperature before examination in the high resolution scanning electron microscopy. Protocols are given on how to collect, to fix, to dehydrate, and to dry plant samples. Subsequently, it is described how to stick them to stubs and cover with a thin conductive layer. These methods are suitable for a wide variety of plant specimens, ranging from microalgae to higher plants.

Fluorescence Lifetime Imaging of Plant Cell Walls.

Fluorescence is a versatile property of many molecules called fluorophores. In plant cell walls, fluorescence is generally attributed to aromatic molecules such as lignin. In contrary to fluorescence intensity, fluorescence lifetime is independent from fluorophore concentration. So mapping fluorescence lifetime of plant cell walls represents a complementary approach to acquire chemical and structural information of cell wall components and interactions.