A Short History of Plant Light Microscopy.

When the microscope was first introduced to scientists in the 17th century, it started a revolution. Suddenly, a whole new world, invisible to the naked eye, was opened to curious explorers. In response to this realization, Nehemiah Grew, an English plant anatomist and physiologist and one of the early microscopists, noted in 1682 "that Nothing hereof remains further to be known, is a Thought not well Calculated". Since Grew made his observations, the microscope has undergone numerous variations, developing from early compound microscopes-hollow metal tubes with a lens on each end-to the modern, sophisticated, out-of-the-box super-resolution microscopes available to researchers today. In this Overview article, I describe these developments and discuss how each new and improved variant of the microscope led to major breakthroughs in the life sciences, with a focus on the plant field. These advances start with Grew's simple and-at the time-surprising realization that plant cells are as complex as animals cells, and that the different parts of the plant body indeed qualify to be called "organs", then move on to the development of the groundbreaking "cell theory" in the mid-19th century and the description of eu- and heterochromatin in the early 20th century, and finish with the precise localization of individual proteins in intact, living cells that we can perform today. Indeed, Grew was right; with ever-increasing resolution, there really does not seem to be an end to what can be explored with a microscope. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.

The Arabidopsis thaliana-Fusarium oxysporum strain 5176 pathosystem: an overview.

Fusarium oxysporum is a soil-borne fungal pathogen of several major food crops. Research on understanding the molecular details of fungal infection and the plant's defense mechanisms against this pathogen has long focused mainly on the tomato-infecting F. oxysporum strains and their specific host plant. However, in recent years, the Arabidopsis thaliana-Fusarium oxysporum strain 5176 (Fo5176) pathosystem has additionally been established to study this plant-pathogen interaction with all the molecular biology, genetic, and genomic tools available for the A. thaliana model system. Work on this system has since produced several new insights, especially with regards to the role of phytohormones involved in the plant's defense response, and the receptor proteins and peptide ligands involved in pathogen detection. Furthermore, work with the pathogenic strain Fo5176 and the related endophytic strain Fo47 has demonstrated the suitability of this system for comparative studies of the plant's specific responses to general microbe- or pathogen-associated molecular patterns. In this review, we highlight the advantages of this specific pathosystem, summarize the advances made in studying the molecular details of this plant-fungus interaction, and point out open questions that remain to be answered.

The Dawn of Plant Molecular Biology: How Three Key Methodologies Paved the Way.

The adoption of Arabidopsis thaliana in the 1980s as a universal plant model finally enabled researchers to adopt and take full advantage of the molecular biology tools and methods developed in the bacterial and animal fields since the early 1970s. It further brought the plant sciences up to speed with other research fields, which had been employing widely accepted model organisms for decades. In parallel with this major development, the concurrent establishment of the plant transformation methodology and the description of the cauliflower mosaic virus (CaMV) 35S promoter enabled scientists to create robust transgenic plant lines for the first time, thereby providing a valuable tool for studying gene function. The ability to create transgenic plants launched the plant biotechnology sector, with Monsanto and Plant Genetic Systems developing the first herbicide- and pest-tolerant plants, initiating a revolution in the agricultural industry. Here I review the major developments over a less than 10-year span and demonstrate how they complemented each other to trigger a revolution in plant molecular biology and launch an era of unprecedented progress for the whole plant field. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.

Brassinosteroids Influence Arabidopsis Hypocotyl Graviresponses through Changes in Mannans and Cellulose.

The force of gravity is a constant environmental factor. Plant shoots respond to gravity through negative gravitropism and gravity resistance. These responses are essential for plants to direct the growth of aerial organs away from the soil surface after germination and to keep an upright posture above ground. We took advantage of the effect of brassinosteroids (BRs) on the two types of graviresponses in Arabidopsis thaliana hypocotyls to disentangle functions of cell wall polymers during etiolated shoot growth. The ability of etiolated Arabidopsis seedlings to grow upward was suppressed in the presence of 24-epibrassinolide (EBL) but enhanced in the presence of brassinazole (BRZ), an inhibitor of BR biosynthesis. These effects were accompanied by changes in cell wall mechanics and composition. Cell wall biochemical analyses, confocal microscopy of the cellulose-specific pontamine S4B dye and cellular growth analyses revealed that the EBL and BRZ treatments correlated with changes in cellulose fibre organization, cell expansion at the hypocotyl base and mannan content. Indeed, a longitudinal reorientation of cellulose fibres and growth inhibition at the base of hypocotyls supported their upright posture whereas the presence of mannans reduced gravitropic bending. The negative effect of mannans on gravitropism is a new function for this class of hemicelluloses. We also found that EBL interferes with upright growth of hypocotyls through their uneven thickening at the base.

Plant science's next top models.

BACKGROUND: Model organisms are at the core of life science research. Notable examples include the mouse as a model for humans, baker's yeast for eukaryotic unicellular life and simple genetics, or the enterobacteria phage λ in virology. Plant research was an exception to this rule, with researchers relying on a variety of non-model plants until the eventual adoption of Arabidopsis thaliana as primary plant model in the 1980s. This proved to be an unprecedented success, and several secondary plant models have since been established. Currently, we are experiencing another wave of expansion in the set of plant models. SCOPE: Since the 2000s, new model plants have been established to study numerous aspects of plant biology, such as the evolution of land plants, grasses, invasive and parasitic plant life, adaptation to environmental challenges, and the development of morphological diversity. Concurrent with the establishment of new plant models, the advent of the 'omics' era in biology has led to a resurgence of the more complex non-model plants. With this review, we introduce some of the new and fascinating plant models, outline why they are interesting subjects to study, the questions they will help to answer, and the molecular tools that have been established and are available to researchers. CONCLUSIONS: Understanding the molecular mechanisms underlying all aspects of plant biology can only be achieved with the adoption of a comprehensive set of models, each of which allows the assessment of at least one aspect of plant life. The model plants described here represent a step forward towards our goal to explore and comprehend the diversity of plant form and function. Still, several questions remain unanswered, but the constant development of novel technologies in molecular biology and bioinformatics is already paving the way for the next generation of plant models.

Complete substitution of a secondary cell wall with a primary cell wall in Arabidopsis.

The bulk of a plant's biomass, termed secondary cell walls, accumulates in woody xylem tissues and is largely recalcitrant to biochemical degradation and saccharification1. By contrast, primary cell walls, which are chemically distinct, flexible and generally unlignified2, are easier to deconstruct. Thus, engineering certain primary wall characteristics into xylem secondary walls would be interesting to readily exploit biomass for industrial processing. Here, we demonstrated that by expressing AP2/ERF transcription factors from group IIId and IIIe in xylem fibre cells of mutants lacking secondary walls, we could generate plants with thickened cell wall characteristics of primary cell walls in the place of secondary cell walls. These unique, newly formed walls displayed physicochemical and ultrastructural features consistent with primary walls and had gene expression profiles illustrative of primary wall synthesis. These data indicate that the group IIId and IIIe AP2/ERFs are transcription factors regulating primary cell wall deposition and could form the foundation for exchanging one cell wall type for another in plants.

Building a plant cell wall at a glance.

Plant cells are surrounded by a strong polysaccharide-rich cell wall that aids in determining the overall form, growth and development of the plant body. Indeed, the unique shapes of the 40-odd cell types in plants are determined by their walls, as removal of the cell wall results in spherical protoplasts that are amorphic. Hence, assembly and remodeling of the wall is essential in plant development. Most plant cell walls are composed of a framework of cellulose microfibrils that are cross-linked to each other by heteropolysaccharides. The cell walls are highly dynamic and adapt to the changing requirements of the plant during growth. However, despite the importance of plant cell walls for plant growth and for applications that we use in our daily life such as food, feed and fuel, comparatively little is known about how they are synthesized and modified. In this Cell Science at a Glance article and accompanying poster, we aim to illustrate the underpinning cell biology of the synthesis of wall carbohydrates, and their incorporation into the wall, in the model plant Arabidopsis.

Antagonistic Transcription Factor Complexes Modulate the Floral Transition in Rice.

Plants measure day or night lengths to coordinate specific developmental changes with a favorable season. In rice (Oryza sativa), the reproductive phase is initiated by exposure to short days when expression of HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1) is induced in leaves. The cognate proteins are components of the florigenic signal and move systemically through the phloem to reach the shoot apical meristem (SAM). In the SAM, they form a transcriptional activation complex with the bZIP transcription factor OsFD1 to start panicle development. Here, we show that Hd3a and RFT1 can form transcriptional activation or repression complexes also in leaves and feed back to regulate their own transcription. Activation complexes depend on OsFD1 to promote flowering. However, additional bZIPs, including Hd3a BINDING REPRESSOR FACTOR1 (HBF1) and HBF2, form repressor complexes that reduce Hd3a and RFT1 expression to delay flowering. We propose that Hd3a and RFT1 are also active locally in leaves to fine-tune photoperiodic flowering responses.

System-wide organization of actin cytoskeleton determines organelle transport in hypocotyl plant cells.

The actin cytoskeleton is an essential intracellular filamentous structure that underpins cellular transport and cytoplasmic streaming in plant cells. However, the system-level properties of actin-based cellular trafficking remain tenuous, largely due to the inability to quantify key features of the actin cytoskeleton. Here, we developed an automated image-based, network-driven framework to accurately segment and quantify actin cytoskeletal structures and Golgi transport. We show that the actin cytoskeleton in both growing and elongated hypocotyl cells has structural properties facilitating efficient transport. Our findings suggest that the erratic movement of Golgi is a stable cellular phenomenon that might optimize distribution efficiency of cell material. Moreover, we demonstrate that Golgi transport in hypocotyl cells can be accurately predicted from the actin network topology alone. Thus, our framework provides quantitative evidence for system-wide coordination of cellular transport in plant cells and can be readily applied to investigate cytoskeletal organization and transport in other organisms.

Studying Protein-Protein Interactions In Planta Using Advanced Fluorescence Microscopy.

The formation of protein complexes through direct protein-protein interaction is essential for virtually every biological process, and accordingly the ability to determine the interaction properties of specific proteins is important to understand these processes. Förster resonance energy transfer (FRET) measurements are state-of-the-art confocal fluorescence microscopy- and imaging-based techniques that allow the analysis of protein interactions in vivo and in planta, in specific compartments of single cells or tissues. Here we provide a step-by-step guide to perform FRET measurements by acceptor photobleaching (APB) and fluorescence lifetime imaging microscopy (FLIM) in the plant expression system Nicotiana benthamiana.

Cell Wall Heterogeneity in Root Development of Arabidopsis.

Plant cell walls provide stability and protection to plant cells. During growth and development the composition of cell walls changes, but provides enough strength to withstand the turgor of the cells. Hence, cell walls are highly flexible and diverse in nature. These characteristics are important during root growth, as plant roots consist of radial patterns of cells that have diverse functions and that are at different developmental stages along the growth axis. Young stem cell daughters undergo a series of rapid cell divisions, during which new cell walls are formed that are highly dynamic, and that support rapid anisotropic cell expansion. Once the cells have differentiated, the walls of specific cell types need to comply with and support different cell functions. For example, a newly formed root hair needs to be able to break through the surrounding soil, while endodermal cells modify their walls at distinct positions to form Casparian strips between them. Hence, the cell walls are modified and rebuilt while cells transit through different developmental stages. In addition, the cell walls of roots readjust to their environment to support growth and to maximize nutrient uptake. Many of these modifications are likely driven by different developmental and stress signaling pathways. However, our understanding of how such pathways affect cell wall modifications and what enzymes are involved remain largely unknown. In this review we aim to compile data linking cell wall content and re-modeling to developmental stages of root cells, and dissect how root cell walls respond to certain environmental changes.

CLAVATA-WUSCHEL signaling in the shoot meristem.

Shoot meristems are maintained by pluripotent stem cells that are controlled by CLAVATA-WUSCHEL feedback signaling. This pathway, which coordinates stem cell proliferation with differentiation, was first identified in Arabidopsis, but appears to be conserved in diverse higher plant species. In this Review, we highlight the commonalities and differences between CLAVATA-WUSCHEL pathways in different species, with an emphasis on Arabidopsis, maize, rice and tomato. We focus on stem cell control in shoot meristems, but also briefly discuss the role of these signaling components in root meristems.

Cellulose-Microtubule Uncoupling Proteins Prevent Lateral Displacement of Microtubules during Cellulose Synthesis in Arabidopsis.

Cellulose is the most abundant biopolymer on Earth and is the major contributor to plant morphogenesis. Cellulose is synthesized by plasma membrane-localized cellulose synthase complexes (CSCs). Nascent cellulose microfibrils become entangled in the cell wall, and further catalysis therefore drives the CSC forward through the membrane: a process guided by cortical microtubules via the protein CSI1/POM2. Still, it is unclear how the microtubules can withstand the forces generated by the motile CSCs to effectively direct CSC movement. Here, we identified a family of microtubule-associated proteins, the cellulose synthase-microtubule uncouplings (CMUs), that located as static puncta along cortical microtubules. Functional disruption of the CMUs caused lateral microtubule displacement and compromised microtubule-based guidance of CSC movement. CSCs that traversed the microtubules interacted with the microtubules via CSI1/POM2, which prompted the lateral microtubule displacement. Hence, we have revealed how microtubules can withstand the propulsion of the CSCs during cellulose biosynthesis and thus sustain anisotropic plant cell growth.

Shared and distinct functions of the pseudokinase CORYNE (CRN) in shoot and root stem cell maintenance of Arabidopsis.

Stem cell maintenance in plants depends on the activity of small secreted signaling peptides of the CLAVATA3/EMBRYO SURROUNDING REGION (CLE) family, which, in the shoot, act through at least three kinds of receptor complexes, CLAVATA1 (CLV1) homomers, CLAVATA2 (CLV2) / CORYNE (CRN) heteromers, and CLV1/CLV2/CRN multimers. In the root, the CLV2/CRN receptor complexes function in the proximal meristem to transmit signals from the CLE peptide CLE40. While CLV1 consists of an extracellular receptor domain and an intracellular kinase domain, CLV2, a leucine-rich repeat (LRR) receptor-like protein, and CRN, a protein kinase, have to interact to form a receptor-kinase complex. The kinase domain of CRN has been reported to be catalytically inactive, and it is not yet known how the CLV2/CRN complex can relay the perceived signal into the cells, and whether the kinase domain is necessary for signal transduction at all. In this study we show that the kinase domain of CRN is actively involved in CLV3 signal transduction in the shoot apical meristem of Arabidopsis, but it is dispensable for CRN protein function in root meristem maintenance. Hence, we provide an example of a catalytically inactive pseudokinase that is involved in two homologous pathways, but functions in distinctively different ways in each of them.