Roles of Reactive Carbonyl Species (RCS) in Plant Response to Abiotic Stress.

Abiotic and biotic stress conditions lead to production of reactive carbonyl species (RCS) which are lipid peroxide derivatives and have detrimental effects on plant cells especially at high concentrations. There are several molecules that can be classified in RCS; among them, 4-hydroxy-(E)-2-nonenal (HNE) and acrolein are widely recognized and studied because of their toxicity. The toxicity mechanisms of RCS are well known in animals but their roles in plant systems especially signaling aspects in metabolism need to be addressed. This chapter focuses on the production mechanisms of RCS in plants as well as how plants scavenge and modify them to prevent irreversible damage in the cell. We aimed to get a comprehensive look at the literature to summarize the signaling roles of RCS in plant metabolism and their interaction with other signaling mechanisms such as highly recognized reactive oxygen species (ROS) signaling. Changing climate promotes more severe abiotic stress effects on plants which also decrease yield on the field. The effects of abiotic stress conditions on RCS metabolism are also gathered in this chapter including their signaling roles during abiotic stresses. Different methods of measuring RCS in plants are also presented in this chapter to draw more attention to the study of RCS metabolism in plants.

Engineering Plant Cell Fates and Functions for Agriculture and Industry.

Many plant species are grown to enable access to specific organs or tissues, such as seeds, fruits, or stems. In some cases, a value is associated with a molecule that accumulates in a single type of cell. Domestication and subsequent breeding have often increased the yields of these target products by increasing the size, number, and quality of harvested organs and tissues but also via changes to overall plant growth architecture to suit large-scale cultivation. Many of the mutations that underlie these changes have been identified in key regulators of cellular identity and function. As key determinants of yield, these regulators are key targets for synthetic biology approaches to engineer new forms and functions. However, our understanding of many plant developmental programs and cell-type specific functions is still incomplete. In this Perspective, we discuss how advances in cellular genomics together with synthetic biology tools such as biosensors and DNA-recording devices are advancing our understanding of cell-specific programs and cell fates. We then discuss advances and emerging opportunities for cell-type-specific engineering to optimize plant morphology, responses to the environment, and the production of valuable compounds.

Metal-Organic Framework-Mediated Delivery of Nucleic Acid across Intact Plant Cells.

Plant synthetic biology is applied in sustainable agriculture, clean energy, and biopharmaceuticals, addressing crop improvement, pest resistance, and plant-based vaccine production by introducing exogenous genes into plants. This technique faces challenges delivering genes due to plant cell walls and intact cell membranes. Novel approaches are required to address this challenge, such as utilizing nanomaterials known for their efficiency and biocompatibility in gene delivery. This work investigates metal-organic frameworks (MOFs) for gene delivery in intact plant cells by infiltration. Hence, small-sized ZIF-8 nanoparticles (below 20 nm) were synthesized and demonstrated effective DNA/RNA delivery into Nicotiana benthamiana leaves and Arabidopsis thaliana roots, presenting a promising and simplified method for gene delivery in intact plant cells. We further demonstrate that small-sized ZIF-8 nanoparticles protect RNA from RNase degradation and successfully silence an endogenous gene by delivering siRNA in N. benthamiana leaves.

Emerging Roles of Extracelluar Vesicles Derived from Bacteria, Mammalian or Plant Cells in the Pathogenesis and Clinical Application of Neurodegenerative Diseases.

A growing number of studies have indicated that extracellular vesicles (EVs), such as exosomes, are involved in the development of neurodegenerative diseases. Components of EVs with biological effects like proteins, nucleic acids, or other molecules can be delivered to recipient cells to mediate physio-/pathological processes. For instance, some aggregate-prone proteins, such as ß-amyloid and α-synuclein, had been found to propagate through exosomes. Therefore, either an increase of detrimental molecules or a decrease of beneficial molecules enwrapped in EVs may fully or partly indicate disease progression. Numerous studies have demonstrated that dysbiosis of the gut microbiota and neurodegeneration are tightly correlated, well-known as the "gut-brain axis". Accumulating evidence has revealed that the gut bacteria-derived EVs play a pivotal role in mediating microbe-host interactions and affect the function of the "gut-brain axis", which subsequently contributes to the pathogenesis of neurodegenerative diseases. In this review, we first briefly discuss the role of EVs from mammalian cells and microbes in mediating the progression of neurodegenerative diseases, and then propose a novel strategy that employs EVs of plants (plant cell-derived exosome-like nanoparticles) for treating neurodegeneration.

A self-regulatory cell-wall-sensing module at cell edges controls plant growth.

Morphogenesis of multicellular organs requires coordination of cellular growth. In plants, cell growth is determined by turgor pressure and the mechanical properties of the cell wall, which also glues cells together. Because plants have to integrate tissue-scale mechanical stresses arising through growth in a fixed tissue topology, they need to monitor cell wall mechanical status and adapt growth accordingly. Molecular factors have been identified, but whether cell geometry contributes to wall sensing is unknown. Here we propose that plant cell edges act as cell-wall-sensing domains during growth. We describe two Receptor-Like Proteins, RLP4 and RLP4-L1, which occupy a unique polarity domain at cell edges established through a targeted secretory transport pathway. We show that RLP4s associate with the cell wall at edges via their extracellular domain, respond to changes in cell wall mechanics and contribute to directional growth control in Arabidopsis.

Plant cell size: Links to cell cycle, differentiation and ploidy.

Cell size affects many processes, including exchange of nutrients and external signals, cell division and tissue mechanics. Across eukaryotes, cells have evolved mechanisms that assess their own size to inform processes such as cell cycle progression or gene expression. Here, we review recent progress in understanding plant cell size regulation and its implications, relating these findings to work in other eukaryotes. Highlights include use of DNA contents as reference point to control the cell cycle in shoot meristems, a size-dependent cell fate decision during stomatal development and insights into the interconnection between ploidy, cell size and cell wall mechanics.

Quantitation of ER Morphology and Dynamics.

The plant endoplasmic reticulum forms a network of tubules connected by three-way junctions or sheet-like cisternae. Although the network is three-dimensional, in many plant cells, it is constrained to thin volume sandwiched between the vacuole and plasma membrane, effectively restricting it to a 2-D planar network. The structure of the network, and the morphology of the tubules and cisternae can be automatically extracted following intensity-independent edge-enhancement and various segmentation techniques to give an initial pixel-based skeleton, which is then converted to a graph representation. ER dynamics can be determined using optical flow techniques from computer vision or persistency analysis. Collectively, this approach yields a wealth of quantitative metrics for ER structure and can be used to describe the effects of pharmacological treatments or genetic manipulation. The software is publicly available.

Using Optical Tweezers Combined with Total Internal Reflection Microscopy to Study Interactions Between the ER and Golgi in Plant Cells.

Optical tweezers have been used to trap and micro-manipulate several biological specimens ranging from DNA, macromolecules, organelles, to single-celled organisms. Using a combination of the refraction and scattering of laser light from a focused laser beam, refractile objects are physically captured and can be moved within the surrounding media. The technique is routinely used to determine biophysical properties such as the forces exerted by motor proteins. Here, we describe how optical tweezers combined with total internal reflection fluorescence microscopy (TIRF) can be used to assess physical interactions between organelles, more specifically the ER and Golgi bodies in plant cells.

Using ER-Targeted Photoconvertible Fluorescent Proteins in Living Plant Cells.

Photoconvertible fluorescent proteins (pcFPs) enable differential coloring of a single organelle. Several pcFP-based probes have been targeted to the endoplasmic reticulum (ER) and can serve as useful tools to study ER dynamics and interactions with other organelles. Here, we describe the procedure to conduct live-cell imaging experiments using ER-targeted pcFP-based probes. Potential problems that might occur during the experiments, their solutions, and several ways to improve the experiments are discussed.

A complex array of factors regulate the activity of Arabidopsis thaliana δ1 -pyrroline-5-carboxylate synthetase isoenzymes to ensure their specific role in plant cell metabolism.

The first and committed step in proline synthesis from glutamate is catalyzed by δ1 -pyrroline-5-carboxylate synthetase (P5CS). Two P5CS genes have been found in most angiosperms, one constitutively expressed to satisfy proline demand for protein synthesis, the other stress-induced. Despite the number of papers to investigate regulation at the transcriptional level, to date, the properties of the enzymes have been subjected to limited study. The isolation of Arabidopsis thaliana P5CS isoenzymes was achieved through heterologous expression and affinity purification. The two proteins were characterized with respect to kinetic and biochemical properties. AtP5CS2 showed KM values in the micro- to millimolar range, and its activity was inhibited by NADP+ , ADP and proline, and by glutamine and arginine at high levels. Mg2+ ions were required for activity, which was further stimulated by K+ and other cations. AtP5CS1 displayed positive cooperativity with glutamate and was almost insensitive to inhibition by proline. In the presence of physiological, nonsaturating concentrations of glutamate, proline was slightly stimulatory, and glutamine strongly increased the catalytic rate. Data suggest that the activity of AtP5CS isoenzymes is differentially regulated by a complex array of factors including the concentrations of proline, glutamate, glutamine, monovalent cations and pyridine dinucleotides.

The selenium-independent phospholipid hydroperoxide glutathione peroxidase from Theobroma cacao (TcPHGPX) protects plant cells against damages and cell death.

Proteins from the glutathione peroxidase (GPX) family, such as GPX4 or PHGPX in animals, are extensively studied for their antioxidant functions and apoptosis inhibition. GPXs can be selenium-independent or selenium-dependent, with selenium acting as a potential cofactor for GPX activity. However, the relationship of plant GPXs to these functions remains unclear. Recent research indicated an upregulation of Theobroma cacao phospholipid hydroperoxide glutathione peroxidase gene (TcPHGPX) expression during early witches' broom disease stages, suggesting the use of antioxidant mechanisms as a plant defense strategy to reduce disease progression. Witches' broom disease, caused by the hemibiotrophic fungus Moniliophthora perniciosa, induces cell death through elicitors like MpNEP2 in advanced infection stages. In this context, in silico and in vitro analyses of TcPHGPX's physicochemical and functional characteristics may elucidate its antioxidant potential and effects against cell death, enhancing understanding of plant GPXs and informing strategies to control witches' broom disease. Results indicated TcPHGPX interaction with selenium compounds, mainly sodium selenite, but without improving the protein function. Protein-protein interaction network suggested cacao GPXs association with glutathione and thioredoxin metabolism, engaging in pathways like signaling, peroxide detection for ABA pathway components, and anthocyanin transport. Tests on tobacco cells revealed that TcPHGPX reduced cell death, associated with decreased membrane damage and H2O2 production induced by MpNEP2. This study is the first functional analysis of TcPHGPX, contributing to knowledge about plant GPXs and supporting studies for witches' broom disease control.

Analysis of Virus-Induced Double-Stranded RNA in Living Plant Cells by the dRBFC Assay.

Double-stranded RNA (dsRNA) is the replicate intermediate of all RNA viruses, and is also recognized by their host cells as a virus-invading molecule signal. Analysis of the localization and dynamic of virus-induced dsRNA can reveal crucial information concerning the molecular mechanism of virus replication and host responses to viral infection. In this chapter, we provide an easy and efficient protocol called dsRNA binding-dependent fluorescence complementation (dRBFC) assay for labeling the dsRNAs in living plant cells using two different plant RNA viruses, namely potato virus X and turnip mosaic virus. Moreover, both YFP- and mRFP-based dRBFC plasmids are available for the flexibility of experiment design.

Green leaf volatiles co-opt proteins involved in molecular pattern signalling in plant cells.

The green leaf volatiles (GLVs) Z-3-hexen-1-ol (Z3-HOL) and Z-3-hexenyl acetate (Z3-HAC) are airborne infochemicals released from damaged plant tissues that induce defenses and developmental responses in receiver plants, but little is known about their mechanism of action. We found that Z3-HOL and Z3-HAC induce similar but distinctive physiological and signaling responses in tomato seedlings and cell cultures. In seedlings, Z3-HAC showed a stronger root growth inhibition effect than Z3-HOL. In cell cultures, the two GLVs induced distinct changes in MAP kinase (MAPK) activity and proton fluxes as well as rapid and massive changes in the phosphorylation status of proteins within 5 min. Many of these phosphoproteins are involved in reprogramming the proteome from cellular homoeostasis to stress and include pattern recognition receptors, a receptor-like cytoplasmic kinase, MAPK cascade components, calcium signaling proteins and transcriptional regulators. These are well-known components of damage-associated molecular pattern (DAMP) signaling pathways. These rapid changes in the phosphoproteome may underly the activation of defense and developmental responses to GLVs. Our data provide further evidence that GLVs function like DAMPs and indicate that GLVs coopt DAMP signaling pathways.

A new method to measure aquaporin-facilitated membrane diffusion of hydrogen peroxide and cations in plant suspension cells.

Plant aquaporins (AQPs) facilitate the membrane diffusion of water and small solutes, including hydrogen peroxide (H2 O2 ) and, possibly, cations, essential signalling molecules in many physiological processes. While the determination of the channel activity generally depends on heterologous expression of AQPs in Xenopus oocytes or yeast cells, we established a genetic tool to determine whether they facilitate the diffusion of H2 O2 through the plasma membrane in living plant cells. We designed genetic constructs to co-express the fluorescent H2 O2 sensor HyPer and AQPs, with expression controlled by a heat shock-inducible promoter in Nicotiana tabacum BY-2 suspension cells. After induction of ZmPIP2;5 AQP expression, a HyPer signal was recorded when the cells were incubated with H2 O2 , suggesting that ZmPIP2;5 facilitates H2 O2 transmembrane diffusion; in contrast, the ZmPIP2;5W85A mutated protein was inactive as a water or H2 O2 channel. ZmPIP2;1, ZmPIP2;4 and AtPIP2;1 also facilitated H2 O2 diffusion. Incubation with abscisic acid and the elicitor flg22 peptide induced the intracellular H2 O2 accumulation in BY-2 cells expressing ZmPIP2;5. We also monitored cation channel activity of ZmPIP2;5 using a novel fluorescent photo-switchable Li+ sensor in BY-2 cells. BY-2 suspension cells engineered for inducible expression of AQPs as well as HyPer expression and the use of Li+ sensors constitute a powerful toolkit for evaluating the transport activity and the molecular determinants of PIPs in living plant cells.

K+ and pH homeostasis in plant cells is controlled by a synchronized K+ /H+ antiport at the plasma and vacuolar membrane.

Stomatal movement involves ion transport across the plasma membrane (PM) and vacuolar membrane (VM) of guard cells. However, the coupling mechanisms of ion transporters in both membranes and their interplay with Ca2+ and pH changes are largely unclear. Here, we investigated transporter networks in tobacco guard cells and mesophyll cells using multiparametric live-cell ion imaging and computational simulations. K+ and anion fluxes at both, PM and VM, affected H+ and Ca2+ , as changes in extracellular KCl or KNO3 concentrations were accompanied by cytosolic and vacuolar pH shifts and changes in [Ca2+ ]cyt and the membrane potential. At both membranes, the K+ transporter networks mediated an antiport of K+ and H+ . By contrast, net transport of anions was accompanied by parallel H+ transport, with differences in transport capacity for chloride and nitrate. Guard and mesophyll cells exhibited similarities in K+ /H+ transport but cell type-specific differences in [H+ ]cyt and pH-dependent [Ca2+ ]cyt signals. Computational cell biology models explained mechanistically the properties of transporter networks and the coupling of transport across the PM and VM. Our integrated approach indicates fundamental principles of coupled ion transport at membrane sandwiches to control H+ /K+ homeostasis and points to transceptor-like Ca2+ /H+ -based ion signaling in plant cells.

Picky eaters: selective autophagy in plant cells.

Autophagy, a fundamental cellular process, plays a vital role in maintaining cellular homeostasis by degrading damaged or unnecessary components. While selective autophagy has been extensively studied in animal cells, its significance in plant cells has only recently gained attention. In this review, we delve into the intriguing realm selective autophagy in plants, with specific focus on its involvement in nutrient recycling, organelle turnover, and stress response. Moreover, recent studies have unveiled the interesting interplay between selective autophagy and epigenetic mechanisms in plants, elucidating the significance of epigenetic regulation in modulating autophagy-related gene expression and finely tuning the selective autophagy process in plants. By synthesizing existing knowledge, this review highlights the emerging field of selective autophagy in plant cells, emphasizing its pivotal role in maintaining nutrient homeostasis, facilitating cellular adaptation, and shedding light on the epigenetic regulation that governs these processes. Our comprehensive study provides the way for a deeper understanding of the dynamic control of cellular responses to nutrient availability and stress conditions, opening new avenues for future research in this field of autophagy in plant physiology.

scPlantDB: a comprehensive database for exploring cell types and markers of plant cell atlases.

Recent advancements in single-cell RNA sequencing (scRNA-seq) technology have enabled the comprehensive profiling of gene expression patterns at the single-cell level, offering unprecedented insights into cellular diversity and heterogeneity within plant tissues. In this study, we present a systematic approach to construct a plant single-cell database, scPlantDB, which is publicly available at https://biobigdata.nju.edu.cn/scplantdb. We integrated single-cell transcriptomic profiles from 67 high-quality datasets across 17 plant species, comprising approximately 2.5 million cells. The data underwent rigorous collection, manual curation, strict quality control and standardized processing from public databases. scPlantDB offers interactive visualization of gene expression at the single-cell level, facilitating the exploration of both single-dataset and multiple-dataset analyses. It enables systematic comparison and functional annotation of markers across diverse cell types and species while providing tools to identify and compare cell types based on these markers. In summary, scPlantDB serves as a comprehensive database for investigating cell types and markers within plant cell atlases. It is a valuable resource for the plant research community.