Live Imaging of the Shoot Apical Meristem of Intact, Soil-Grown, Flowering Arabidopsis Plants.

All aerial organs in plants originate from the shoot apical meristem, a specialized tissue at the tip of a plant, enclosing a few stem cells. Understanding developmental dynamics within this tissue in relation to internal and external stimuli is of crucial importance. Imaging the meristem at the cellular level beyond very early stages requires the apex to be detached from the plant body, a procedure that does not allow studies in living, intact plants over longer periods. This protocol describes a new confocal microscopy method with the potential to image the shoot apical meristem of an intact, soil-grown, flowering Arabidopsis plant over several days. The setup opens new avenues to study apical stem cells, their interconnection with the whole plant, and their responses to environmental stimuli. Key features • Novel dissection and imaging method of the shoot apical meristem of Arabidopsis. • Procedure performed with intact, soil-grown, flowering plants. • Possibility of long-term live imaging of the shoot apical meristem. • Protocol can be adapted to different plant species.

Cytokinins - regulators of de novo shoot organogenesis.

Plants, unlike animals, possess a unique developmental plasticity, that allows them to adapt to changing environmental conditions. A fundamental aspect of this plasticity is their ability to undergo postembryonic de novo organogenesis. This requires the presence of regulators that trigger and mediate specific spatiotemporal changes in developmental programs. The phytohormone cytokinin has been known as a principal regulator of plant development for more than six decades. In de novo shoot organogenesis and in vitro shoot regeneration, cytokinins are the prime candidates for the signal that determines shoot identity. Both processes of de novo shoot apical meristem development are accompanied by changes in gene expression, cell fate reprogramming, and the switching-on of the shoot-specific homeodomain regulator, WUSCHEL. Current understanding about the role of cytokinins in the shoot regeneration will be discussed.

3D imaging reveals apical stem cell responses to ambient temperature.

Plant growth is driven by apical meristems at the shoot and root growth points, which comprise continuously active stem cell populations. While many of the key factors involved in homeostasis of the shoot apical meristem (SAM) have been extensively studied under artificial constant growth conditions, only little is known how variations in the environment affect the underlying regulatory network. To shed light on the responses of the SAM to ambient temperature, we combined 3D live imaging of fluorescent reporter lines that allowed us to monitor the activity of two key regulators of stem cell homeostasis in the SAM namely CLAVATA3 (CLV3) and WUSCHEL (WUS), with computational image analysis to derive morphological and cellular parameters of the SAM. Whereas CLV3 expression marks the stem cell population, WUS promoter activity is confined to the organizing center (OC), the niche cells adjacent to the stem cells, hence allowing us to record on the two central cell populations of the SAM. Applying an integrated computational analysis of our data we found that variations in ambient temperature not only led to specific changes in spatial expression patterns of key regulators of SAM homeostasis, but also correlated with modifications in overall cellular organization and shoot meristem morphology.

[Construction strategies and prospect of the Global Medicinal Plant Stem Cell Bank].

Medicinal plant stem cells are separated from the meristem and vascular cambium of medicinal plants, which can produce active components for preventing and treating diseases and improving body physical functions under certain conditions. Medicinal plant stem cells come from a broad category of medicinal plants, including ethnic medicinal plants, folk medicinal plants, original plants of health products, vegetables, fruits, and other potential medicinal plants. At present, the techniques for the isolation, identification, preservation and culture of medicinal plant stem cells have become increasingly mature, and the mechanism of stem cell differentiation, growth and regulation of secondary metabolites has been studied in depth. Medicinal plant stem cells have a broad application prospect in medicine, health food, food and medical beauty products. As a strategic resource, the construction of the "Global Medicinal Plant Stem Cell Bank" was first proposed to preserve various kinds of medicinal plant resources in the world, and it will go global relying on the internationalization strategy of traditional Chinese medicine. The bank should follow safety, environmental protection, advanced and practical design principles. The main construction contents include the original plant bank, stem cell bank, component resource bank, gene bank, database and resource sharing system, with genetic and data resources incorporated into the scope of protection and utilization. The bank will establish a new strategy for medicinal plant resources protection and regeneration, and provide a new resource for natural products drug discovery and a technology sharing platform for various medicinal plant stem cells. As a resource treasury, a source of innovative technologies and a center of cooperation, it will become the core driving force of the global medicinal plant stem cell industry.

The intrinsic chaperone network of Arabidopsis stem cells confers protection against proteotoxic stress.

The biological purpose of plant stem cells is to maintain themselves while providing new pools of differentiated cells that form organs and rejuvenate or replace damaged tissues. Protein homeostasis or proteostasis is required for cell function and viability. However, the link between proteostasis and plant stem cell identity remains unknown. In contrast to their differentiated counterparts, we find that root stem cells can prevent the accumulation of aggregated proteins even under proteotoxic stress conditions such as heat stress or proteasome inhibition. Notably, root stem cells exhibit enhanced expression of distinct chaperones that maintain proteome integrity. Particularly, intrinsic high levels of the T-complex protein-1 ring complex/chaperonin containing TCP1 (TRiC/CCT) complex determine stem cell maintenance and their remarkable ability to suppress protein aggregation. Overexpression of CCT8, a key activator of TRiC/CCT assembly, is sufficient to ameliorate protein aggregation in differentiated cells and confer resistance to proteotoxic stress in plants. Taken together, our results indicate that enhanced proteostasis mechanisms in stem cells could be an important requirement for plants to persist under extreme environmental conditions and reach extreme long ages. Thus, proteostasis of stem cells can provide insights to design and breed plants tolerant to environmental challenges caused by the climate change.

Fundamental mechanisms of the stem cell regulation in land plants: lesson from shoot apical cells in bryophytes.

KEY MESSAGE: This review compares the molecular mechanisms of stem cell control in the shoot apical meristems of mosses and angiosperms and reveals the conserved features and evolution of plant stem cells. The establishment and maintenance of pluripotent stem cells in the shoot apical meristem (SAM) are key developmental processes in land plants including the most basal, bryophytes. Bryophytes, such as Physcomitrium (Physcomitrella) patens and Marchantia polymorpha, are emerging as attractive model species to study the conserved features and evolutionary processes in the mechanisms controlling stem cells. Recent studies using these model bryophyte species have started to uncover the similarities and differences in stem cell regulation between bryophytes and angiosperms. In this review, we summarize findings on stem cell function and its regulation focusing on different aspects including hormonal, genetic, and epigenetic control. Stem cell regulation through auxin, cytokinin, CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) signaling and chromatin modification by Polycomb Repressive Complex 2 (PRC2) and PRC1 is well conserved. Several transcription factors crucial for SAM regulation in angiosperms are not involved in the regulation of the SAM in mosses, but similarities also exist. These findings provide insights into the evolutionary trajectory of the SAM and the fundamental mechanisms involved in stem cell regulation that are conserved across land plants.

Single-cell profiling lights different cell trajectories in plants.

The molecular mechanism of the maintenance and differentiation of plant stem cells is an eternal theme in studies on plant growth and development. Recent advances in single-cell RNA sequencing (scRNA-seq) methods have completely changed the understanding of cell heterogeneity and cell function, allowing research precision to identify the differentiation trajectory of stem cells maintained and differentiated at the cellular level. This review aimed to mainly discuss the novel insights provided by scRNA-seq for the maintenance and initiation of plant stem cells, cell differentiation, cell response to environmental changes, and improvement strategies for scRNA-seq. In addition, it highlighted additional perspectives beyond scRNA-seq, such as spatial transcriptomes, epigenomes, and single-cell multiomics, for a renewed understanding of stem cell maintenance and cell differentiation, thus providing potential targets and theoretical foundations for crop improvement.

Increased Induction of Apoptosis in ESCC (Esophageal Squamous-Cell Carcinoma) by Betula pendula Roth Stem Cell Extract Containing Triterpenoids Compared to Doxorubicin.

BACKGROUND: Esophageal Squamous-Cell Carcinoma (ESCC) is one of the most life-threatening malignancies worldwide, with a growing incidence in Iran higher than the global average. OBJECTIVE: The present study, for the first time under patent number (97668), introduces a method using in vitro production of activated-Birch stem cells using biotechnological techniques of tissue culture and plant stem cell culture from Betula pendula Roth (Birch) bark. METHODS: In the first step, Birch stem cells were produced in large amounts using tissue culture, and then the amount of triterpenoids of its extract was measured by the HPLC method. In the second step, the cytotoxicity was evaluated by MTT, and the IC50 was calculated. The cellular apoptosis in response to the extract compared to doxorubicin was measured using the Annexin V kit and the flow cytometry method. RESULTS: The optimized method introduced in the current study efficiently produced plant stem cells containing triterpenoids in large quantities over a period of 2-4 months. Our findings indicated that the growth of ESCC cells decreased by induction treatment 3 times (24, 36, 48 hours). IC50 values were obtained in 24 hours for the natural bark extract, Birch stem cell extract, doxorubicin and interactions of two extracts with doxorubicin at 300µg/mL, 1700µg/mL, 0.5µM, 150µg/mL, 1800µg/mL, respectively. In the flow cytometric test, the Birch stem cell extract showed the highest percentage of apoptosis, with 92.5% for total apoptosis. The percentage of total apoptosis in doxorubicin treatment was 85.33%, and the combination of doxorubicin with Birch stem cell extract was 88.33%. Natural bark extract and its combination with a lower percentage (69.33% and 70.33%, respectively) caused apoptosis of esophageal cancer cells. CONCLUSION: Owing to the extinction of Birch in Iran and its inaccessibility and exploitation, Birch stem cells can be cultured as an appropriate alternative source to produce valuable triterpenoids for pharmaceutical purposes. Additionally, according to the results of this study, stem cells can be used to enhance the treatment of esophageal cancer and supplementation with chemotherapy.

Plant stem cells and their applications: special emphasis on their marketed products.

Stem cells are becoming increasingly popular in public lexicon owing to their prospective applications in the biomedical and therapeutic domains. Extensive research has found various independent stem cell systems fulfilling specific needs of plant development. Plant stem cells are innately undifferentiated cells present in the plant's meristematic tissues. Such cells have various commercial uses, wherein cosmetic manufacture involving stem cell derivatives is the most promising field at present. Scientific evidence suggests anti-oxidant and anti-inflammatory properties possessed by various plants such as grapes (Vitis vinifera), lilacs (Syringa vulgaris), Swiss apples (Uttwiler spatlauber) etc. are of great importance in terms of cosmetic applications of plant stem cells. There are widespread uses of plant stem cells and their extracts. The products so formulated have a varied range of applications which included skin whitening, de-tanning, moisturizing, cleansing etc. Despite all the promising developments, the domain of plant stem cells remains hugely unexplored. This article presents an overview of the current scenario of plant stem cells and their applications in humans.

Role of chromatin modification and remodeling in stem cell regulation and meristem maintenance in Arabidopsis.

In higher plants, pluripotent stem cells reside in the specialized microenvironment called stem cell niches (SCNs) harbored at the shoot apical meristem (SAM) and root apical meristem (RAM), which give rise to the aerial and underground parts of a plant, respectively. The model plant Arabidopsis thaliana (Arabidopsis) has been extensively studied to decipher the intricate regulatory mechanisms involving some key transcriptions factors and phytohormones that play pivotal roles in stem cell homeostasis, meristem maintenance, and organ formation. However, there is increasing evidence to show the epigenetic regulation of the chromatin architecture, gene expression exerting an influence on an innate balance between the self-renewal of stem cells, and differentiation of the progeny cells to a specific tissue type or organ. Post-translational histone modifications, ATP-dependent chromatin remodeling, and chromatin assembly/disassembly are some of the key features involved in the modulation of chromatin architecture. Here, we discuss the major epigenetic regulators and illustrate their roles in the regulation of stem cell activity, meristem maintenance, and related organ patterning in Arabidopsis.

Quantitative Early Auxin Root Proteomics Identifies GAUT10, a Galacturonosyltransferase, as a Novel Regulator of Root Meristem Maintenance.

Auxin induces rapid gene expression changes throughout root development. How auxin-induced transcriptional responses relate to changes in protein abundance is not well characterized. This report identifies early auxin responsive proteins in roots at 30 min and 2 h after hormone treatment using a quantitative proteomics approach in which 3,514 proteins were reliably quantified. A comparison of the >100 differentially expressed proteins at each the time point showed limited overlap, suggesting a dynamic and transient response to exogenous auxin. Several proteins with established roles in auxin-mediated root development exhibited altered abundance, providing support for this approach. While novel targeted proteomics assays demonstrate that all six auxin receptors remain stable in response to hormone. Additionally, 15 of the top responsive proteins display root and/or auxin response phenotypes, demonstrating the validity of these differentially expressed proteins. Auxin signaling in roots dictates proteome reprogramming of proteins enriched for several gene ontology terms, including transcription, translation, protein localization, thigmatropism, and cell wall modification. In addition, we identified auxin-regulated proteins that had not previously been implicated in auxin response. For example, genetic studies of the auxin responsive protein galacturonosyltransferase 10 demonstrate that this enzyme plays a key role in root development. Altogether these data complement and extend our understanding of auxin response beyond that provided by transcriptome studies and can be used to uncover novel proteins that may mediate root developmental programs.

Plant stem cells: what we know and what is anticipated.

Plant stem cell research is of interest due to stem cells ability of unlimited division, therapeutic potential and steady supply to provide precursor cells. Their isolation and culture provides the important source for the production of homogenous lines of active constituents that allow large-scale production of various metabolites. The process of dedifferentiation and reversal to pluripotent cells involves the various pathways genes related to the stem cells and are associated to each other for maintaining a specific niche. Domains such as niche dynamics and maintenance signaling can be used for the identification of genes for stem cell niche. Significant findings have been achieved in the past on plant stem cells however our understanding towards mechanisms underlying some specific phenomenon like dedifferentiation, regulation, niche dynamics is still in infancy. The present review is based on the past research efforts and also pave a way forward for the future anticipation in the field of development of cell cultures for the production of active metabolites on large scale and undertanding transcriptional regulation of stem cell genes involved in niche signaling.

Regulation of Division and Differentiation of Plant Stem Cells.

A major challenge in developmental biology is unraveling the precise regulation of plant stem cell maintenance and the transition to a fully differentiated cell. In this review, we highlight major themes coordinating the acquisition of cell identity and subsequent differentiation in plants. Plant cells are immobile and establish position-dependent cell lineages that rely heavily on external cues. Central players are the hormones auxin and cytokinin, which balance cell division and differentiation during organogenesis. Transcription factors and miRNAs, many of which are mobile in plants, establish gene regulatory networks that communicate cell position and fate. Small peptide signaling also provides positional cues as new cell types emerge from stem cell division and progress through differentiation. These pathways recruit similar players for patterning different organs, emphasizing the modular nature of gene regulatory networks. Finally, we speculate on the outstanding questions in the field and discuss how they may be addressed by emerging technologies.

Plant cell culture as emerging technology for production of active cosmetic ingredients.

Plants have always been the main source for active cosmetic ingredients, having proven health beneficial effects on human, such as anti-aging, antioxidant, anti-inflammatory, UV-protective, anti-cancer, anti-wrinkle, skin soothing, whitening, moisturizing, etc. Extracts from herbal, aromatic and/or medicinal plants have been widely used as effective active ingredients in cosmeceuticals or nutricosmetics, especially in products for topical application and skin-care formulations. However, over the past decade, there has been an increasing interest to plant cell culture - derived active cosmetic ingredients. These are "new generation" of high quality natural products, produced by the modern plan biotechnology methods, which usually showed stronger activities than the plant extracts obtained by the classical methods. In this review, the advantages and the current progress in plant cell culture technology for the production of active cosmetic ingredients have been summarized, and discussed in details within a presented case study for calendula stem cell product development.

Plant stem cells in cosmetics: current trends and future directions.

Plant regeneration at the cellular and tissue level is a unique process. Similar to animals, the stem cells in plants have properties that help stimulate and regenerate plants after injury. The unique properties of plant stem cells have been a recent area of interest and focus both in developing new cosmetics and studying how these extracts/phytohormones will influence animal skin. This special report focuses on the current evidence-based trends in plant stem cell-based cosmetics and sheds light on the challenges that we need to overcome in order to see meaningful changes in human skin using topical cosmetics derived from plant stem cells.

Two-Step Functional Innovation of the Stem-Cell Factors WUS/WOX5 during Plant Evolution.

WUS and WOX5, which are expressed, respectively, in the organizing center (OC) and the quiescent center (QC), are essential for shoot/root apical stem-cell maintenance in flowering plants. However, little is known about how these stem-cell factors evolved their functions in flowering plants. Here, we show that the WUS/WOX5 proteins acquired two distinct capabilities by a two-step functional innovation process in the course of plant evolution. The first-step is the apical stem-cell maintenance activity of WUS/WOX5, which originated in the common ancestor of ferns and seed plants, as evidenced by the interspecies complementation experiments, showing that ectopic expression of fern Ceratopteris richardii WUS-like (CrWUL) surrounding OC/QC, or exclusive OC-/QC-expressed gymnosperms/angiosperms WUS/WOX5 in Arabidopsis wus-1 and wox5-1 mutants, could rescue their phenotypes. The second-step is the intercellular mobility that emerged in the common ancestor of seed plants after divergence from the ferns. Evidence for this includes confocal imaging of GFP fusion proteins, showing that WUS/WOX5 from seed plants, rather than from the fern CrWUL, can migrate into cells adjacent to the OC/QC. Evolutionary analysis showed that the WUS-like gene was duplicated into two copies prior to the divergence of gymnosperms/angiosperms. Then the two gene copies (WUS and WOX5) have undergone similar levels of purifying selection, which is consistent with their conserved functions in angiosperm shoot/root stem-cell maintenance and floral organ formation. Our results highlight the critical roles and the essential prerequisites that the two-step functional innovation of these genes performs and represents in the origin of flowering plants.

Cell size and growth regulation in the Arabidopsis thaliana apical stem cell niche.

Cell size and growth kinetics are fundamental cellular properties with important physiological implications. Classical studies on yeast, and recently on bacteria, have identified rules for cell size regulation in single cells, but in the more complex environment of multicellular tissues, data have been lacking. In this study, to characterize cell size and growth regulation in a multicellular context, we developed a 4D imaging pipeline and applied it to track and quantify epidermal cells over 3-4 d in Arabidopsis thaliana shoot apical meristems. We found that a cell size checkpoint is not the trigger for G2/M or cytokinesis, refuting the unexamined assumption that meristematic cells trigger cell cycle phases upon reaching a critical size. Our data also rule out models in which cells undergo G2/M at a fixed time after birth, or by adding a critical size increment between G2/M transitions. Rather, cell size regulation was intermediate between the critical size and critical increment paradigms, meaning that cell size fluctuations decay by ∼75% in one generation compared with 100% (critical size) and 50% (critical increment). Notably, this behavior was independent of local cell-cell contact topologies and of position within the tissue. Cells grew exponentially throughout the first >80% of the cell cycle, but following an asymmetrical division, the small daughter grew at a faster exponential rate than the large daughter, an observation that potentially challenges present models of growth regulation. These growth and division behaviors place strong constraints on quantitative mechanistic descriptions of the cell cycle and growth control.

Ascorbate oxidase is the potential conductor of a symphony of signaling pathways.

The functional role of ascorbate oxidase (AO; EC 1.10.3.3) has never been fully explained so far, due to the difficulties in understanding the presence of an enzyme specifically oxidizing ascorbate with no obvious advantage, and the apparent disadvantage of lowering plant stress resistance as a consequence of ascorbate consumption. Here we suggest a complete change of perspective, by proposing an essential role of AO as a modulator of both ascorbate and oxygen content, with relevant implications related to signaling. By affecting the overall redox state, AO is actually involved in redox regulation in the extracellular matrix. In addition, AO can contribute to creating a hypoxic microenvironment, especially relevant in the maintenance of meristem identity and the establishment of mutualistic plant-microbe interactions. We also hypothesize the possible involvement of AO in the activation of a signaling cascade analogous to the mechanism of prolyl hydroxylases/Hypoxia Inducible Factors in animals.