Living Plant Cells Research Articles

Changes in Cytosolic ATP Levels and Intracellular Morphology during Bacteria-Induced Hypersensitive Cell Death as Revealed by Real-Time Fluorescence Microscopy Imaging

Hypersensitive cell death is known to involve dynamic remodeling of intracellular structures that uses energy released during ATP hydrolysis. However, the relationship between intracellular structural changes and ATP levels during hypersensitive cell death remains unclear. Here, to visualize ATP dynamics directly in real time in individual living plant cells, we applied a genetically encoded Förster resonance energy transfer (FRET)-based fluorescent ATP indicator, ATeam1.03-nD/nA, for plant cells. Intracellular ATP levels increased approximately 3 h after inoculation with the avirulent strain DC3000/avrRpm1 of Pseudomonas syringae pv. tomato (Pst), which was accompanied by the simultaneous disappearance of transvacuolar strands and appearance of bulb-like structures within the vacuolar lumen. Approximately 5 h after bacterial inoculation, the bulb-like structures disappeared and ATP levels drastically decreased. After another 2 h, the large central vacuole was disrupted. In contrast, no apparent changes in intracellular ATP levels were observed in the leaves inoculated with the virulent strain Pst DC3000. The Pst DC3000/avrRpm1-induced hypersensitive cell death was strongly suppressed by inhibiting ATP synthesis after oligomycin A application within 4 h after bacterial inoculation. When the inhibitor was applied 7 h after bacterial inoculation, cell death was unaffected. These observations show that changes in intracellular ATP levels correlate with intracellular morphological changes during hypersensitive cell death, and that ATP is required just before vacuolar rupture in response to bacterial infection.

In planta localization and interactions of impatiens necrotic spot tospovirus proteins

Impatiens necrotic spot tospovirus (INSV) is a significant pathogen of ornamentals. The tripartite negative- and ambi-sense RNA genome encodes six proteins that are involved in cytoplasmic replication, movement, assembly, insect transmission and defence. To gain insight into the associations of these viral proteins, we determined their intracellular localization and interactions in living plant cells. Nucleotide sequences encoding the nucleoprotein N, non-structural proteins NSs and NSm, and glycoproteins Gn and Gc of a Kentucky isolate of INSV were amplified by RT-PCR, cloned, sequenced and transiently expressed as fusions with autofluorescent proteins in leaf epidermal cells of Nicotiana benthamiana. All proteins accumulated at the cell periphery and co-localized with an endoplasmic reticulum marker. The Gc protein fusion also localized to the nucleus. N and NSm protein self-interactions and an NSm-N interaction were observed by using bimolecular fluorescence complementation. A tospovirus NSm homotypic interaction had not been reported previously.

Screening for in planta protein-protein interactions combining bimolecular fluorescence complementation with flow cytometry

Understanding protein and gene function requires identifying interaction partners using biochemical, molecular or genetic tools. In plants, searching for novel protein-protein interactions is limited to protein purification assays, heterologous in vivo systems such as the yeast-two-hybrid or mutant screens. Ideally one would be able to search for novel protein partners in living plant cells. We demonstrate that it is possible to screen for novel protein-protein interactions from a random library in protoplasted Arabidopsis plant cells and recover some of the interacting partners. Our screen is based on capturing the bi-molecular complementation of mYFP between an YN-bait fusion partner and a completely random prey YC-cDNA library with FACS. The candidate interactions were confirmed using in planta BiFC assays and in planta FRET-FLIM assays. From this work, we show that the well characterized protein Calcium Dependent Protein Kinase 3 (CPK3) interacts with APX3, HMGB5, ORP2A and a ricin B-related lectin domain containing protein At2g39050. This is one of the first randomin planta screens to be successfully employed.

The Dynamics of Changes in Starch and Lipid Droplets and Sub-Cellular Localization of β-Amylase During the Growth of Lily Bulbs

The ultra-structure of mother and outer daughter scales of Lilium Oriental hybrid Sorbonne were studied using transmission electron microscope to examine the sub-cellular localization of starch and lipid droplets during growth and development from shoot emergence to senescence. The contents of starch granules and lipid droplets in the cell of the mother scales decreased significantly from shoot emergence to anthesis, indicating that these scales served as a source for growth and development. After flowering, the number of starch granules and lipid droplets increased dramatically, and finally the cells were filled with the above molecules indicating that the bulb becomes a major sink during bulb enlargement. Ultrastructure observation also showed that symplastic pathway is the main pathway in cells in the exchange and transportation of material during bulb development. The activity of β-amylase, one of the key enzymes catalyzing starch breakdown, showed a similar trend. The enzyme sub-cellular localization via immune-gold electron-microscopy showed that β-amylase was predominantly located together with starch granules, while the gold particles were scarcely found in other sub-cellular compartments. The result suggested that this enzyme is compartmented together with its functional substrate supporting its function in catalyzing starch breakdown in living plant cells.

A promising new wavelength region for three‐photon fluorescence microscopy of live cells

We report three-photon laser scanning microscopy (3PLSM) using a bi-directional pumped optical parametric oscillator (OPO) with signal wavelength output at λ= 1500 nm. This novel laser was used to overcome the high optical loss in the infrared spectral region observed in laser scanning microscopes and objective lenses that renders them otherwise difficult to use for imaging. To test our system, we performed 3PLSM auto-fluorescence imaging of live plant cells at λ= 1500 nm, specifically Spirogyra, and compared performance with two-photon excitation (2PLSM) imaging using a femtosecond pulsed Ti:Sapphire laser at λ= 780 nm. Analysis of cell viability based on cytoplasmic organelle streaming and structural changes of cells revealed that at similar peak powers, 2PLSM caused gross cell damage after 5 min but 3PLSM showed little or no interference with cell function after 15 min. The λ= 1500 nm OPO is thus shown to be a practical laser source for live cell imaging.

Cytoplasmic streaming in plant cells: the role of wall slip

We present a computer simulation study, via lattice Boltzmann simulations, of a microscopic model for cytoplasmic streaming in algal cells such as those of Chara corallina. We modelled myosin motors tracking along actin lanes as spheres undergoing directed motion along fixed lines. The sphere dimension takes into account the fact that motors drag vesicles or other organelles, and, unlike previous work, we model the boundary close to which the motors move as walls with a finite slip layer. By using realistic parameter values for actin lane and myosin density, as well as for endoplasmic and vacuole viscosity and the slip layer close to the wall, we find that this simplified view, which does not rely on any coupling between motors, cytoplasm and vacuole other than that provided by viscous Stokes flow, is enough to account for the observed magnitude of streaming velocities in intracellular fluid in living plant cells.

Erratum to: Probing and tracking organelles in living plant cells

Erratum to: ProtoplasmaDOI: 10.1007/s00709-011-0364-4TheaffiliationofJozef Samajunfortunatelycontainsamistake.It should have been,Centre of the Region Hana for Biotechnological andAgricultural Research,Department of Cell Biology, Faculty of Science,Palacký University Olomouc,Slechtitelu 11, 78371 Olomouc, Czech Republic

Viscoelastic properties of cell walls of single living plant cells determined by dynamic nanoindentation

Plant development results from controlled cell divisions, structural modifications, and reorganizations of the cell wall. Thereby, regulation of cell wall behaviour takes place at multiple length scales involving compositional and architectural aspects in addition to various developmental and/or environmental factors. The physical properties of the primary wall are largely determined by the nature of the complex polymer network, which exhibits time-dependent behaviour representative of viscoelastic materials. Here, a dynamic nanoindentation technique is used to measure the time-dependent response and the viscoelastic behaviour of the cell wall in single living cells at a micron or sub-micron scale. With this approach, significant changes in storage (stiffness) and loss (loss of energy) moduli are captured among the tested cells. The results reveal hitherto unknown differences in the viscoelastic parameters of the walls of same-age similarly positioned cells of the Arabidopsis ecotypes (Col 0 and Ws 2). The technique is also shown to be sensitive enough to detect changes in cell wall properties in cells deficient in the activity of the chromatin modifier ATX1. Extensive computational modelling of the experimental measurements (i.e. modelling the cell as a viscoelastic pressure vessel) is used to analyse the influence of the wall thickness, as well as the turgor pressure, at the positions of our measurements. By combining the nanoDMA technique with finite element simulations quantifiable measurements of the viscoelastic properties of plant cell walls are achieved. Such techniques are expected to find broader applications in quantifying the influence of genetic, biological, and environmental factors on the nanoscale mechanical properties of the cell wall.

Genome modifications in plant cells by custom‐made restriction enzymes

Genome editing, i.e. the ability to mutagenize, insert, delete and replace sequences, in living cells is a powerful and highly desirable method that could potentially revolutionize plant basic research and applied biotechnology. Indeed, various research groups from academia and industry are in a race to devise methods and develop tools that will enable not only site-specific mutagenesis but also controlled foreign DNA integration and replacement of native and transgene sequences by foreign DNA, in living plant cells. In recent years, much of the progress seen in gene targeting in plant cells has been attributed to the development of zinc finger nucleases and other novel restriction enzymes for use as molecular DNA scissors. The induction of double-strand breaks at specific genomic locations by zinc finger nucleases and other novel restriction enzymes results in a wide variety of genetic changes, which range from gene addition to the replacement, deletion and site-specific mutagenesis of endogenous and heterologous genes in living plant cells. In this review, we discuss the principles and tools for restriction enzyme-mediated gene targeting in plant cells, as well as their current and prospective use for gene targeting in model and crop plants.

Dynamics of endoplasmic reticulum during freezing in living plant cells

Dynamics of endoplasmic reticulum during freezing in living plant cells

Probing and tracking organelles in living plant cells

Intracellular organelle movements and positioning play pivotal roles in enabling plants to proliferate life efficiently and to survive diverse environmental stresses. The elaborate dissection of organelle dynamics and their underlying mechanisms (e.g., the role of the cytoskeleton in organelle movements) largely depends on the advancement and efficiency of organelle tracking systems. Here, we provide an overview of some recently developed tools for labeling and tracking organelle dynamics in living plant cells.

Dynamic behavior of clathrin in Arabidopsis thaliana unveiled by live imaging

Clathrin-coated vesicles (CCV) are necessary for selective transport events, including receptor-mediated endocytosis on the plasma membrane and cargo molecule sorting in the trans-Golgi network (TGN). Components involved in CCV formation include clathrin heavy and light chains and several adaptor proteins that are conserved among plants. Clathrin-dependent endocytosis has been shown to play an integral part in plant endocytosis. However, little information is known about clathrin dynamics in living plant cells. In this study, we have visualized clathrin in Arabidopsis thaliana by tagging clathrin light chain with green fluorescent protein (CLC-GFP). Quantitative evaluations of colocalization demonstrate that the majority of CLC-GFP is localized to the TGN, and a minor population is associated with multivesicular endosomes and the Golgi trans-cisternae. Live imaging further demonstrated the presence of highly dynamic clathrin-positive tubules and vesicles, which appeared to mediate interactions between the TGNs. CLC-GFP is also targeted to cell plates and the plasma membrane. Although CLC-GFP colocalizes with a dynamin isoform at the plasma membrane, these proteins exhibit distinct distributions at newly forming cell plates. This finding indicates independent functions of CLC (clathrin light chains) and dynamin during the formation of cell plates. We have also found that brefeldin A and wortmannin treatment causes distinctly different alterations in the dynamics and distribution of clathrin-coated domains at the plasma membrane. This could account for the different effects of these drugs on plant endocytosis.

CBS Domain Proteins Regulate Redox Homeostasis

fluorescent protein fusion localized to the chloroplast. In the chloroplast, CBSX1 interacted with four distinct thioredoxins, as shown by yeast two-hybrid and in vitro pull-down studies. The interaction of CBSX1 and thioredoxin also occurred in living plant cells, as shown by bimolecular fluorescence complementation analysis in tobacco (Nicotiana tabacum) leaves. Based on these interactions, the authors hypothesize that CBSX1 and CBX2 positively regulate thioredoxins, which then reduce target proteins, such as peroxiredoxin, which in turn reduce cellular H2O2 and regulate its level. They show that both CBSX proteins increase the activity of all four thioredoxins, in some cases more effectively than the chemical reductant DTT. Addition of AMP further increased the activation of all four thioredoxins by CBSX1 and CBSX2, but ADP and ATP did not have this enhancing effect. The authors also present a crystal structure of CBSX2 and describe a positively charged ligand binding pocket formed by Lys and Arg residues. This pocket is similar to those in other CBS domain proteins that contain adenosinerelated ligands. In CBSX1, this pocket can accommodate AMP, but not the bulkier ADP or ATP, consistent with the above demonstration that CBSX1induced activation of thioredoxin was en

Anti-Apoptotic Machinery Protects the Necrotrophic Fungus Botrytis cinerea from Host-Induced Apoptotic-Like Cell Death during Plant Infection

Necrotrophic fungi are unable to occupy living plant cells. How such pathogens survive first contact with living host tissue and initiate infection is therefore unclear. Here, we show that the necrotrophic grey mold fungus Botrytis cinerea undergoes massive apoptotic-like programmed cell death (PCD) following germination on the host plant. Manipulation of an anti-apoptotic gene BcBIR1 modified fungal response to PCD-inducing conditions. As a consequence, strains with reduced sensitivity to PCD were hyper virulent, while strains in which PCD was over-stimulated showed reduced pathogenicity. Similarly, reduced levels of PCD in the fungus were recorded following infection of Arabidopsis mutants that show enhanced susceptibility to B. cinerea. When considered together, these results suggest that Botrytis PCD machinery is targeted by plant defense molecules, and that the fungal anti-apoptotic machinery is essential for overcoming this host-induced PCD and hence, for establishment of infection. As such, fungal PCD machinery represents a novel target for fungicides and antifungal drugs.

Förster resonance energy transfer demonstrates a flavonoid metabolon in living plant cells that displays competitive interactions between enzymes

We have used Förster resonance energy transfer detected by fluorescence lifetime imaging microscopy (FLIM-FRET) to provide the first evidence from living plants cells for the existence of a flavonoid metabolon. The distribution of flux within this system may be regulated by the direct competition of enzymes that catalyze key branch-point reactions, flavonol synthase 1 and dihydroflavonol 4-reductase, for association with the entry-point enzyme, chalcone synthase. Because the flavonoid enzymes were likely recruited from pathways of primary metabolism, our findings suggest a new general working model for the regulation of dynamic pathways in their native cellular context. Structured summary of protein interactionsCHS and FLS1physically interact by fluorescence resonance energy transfer(View interaction)CHS and DFRphysically interact by fluorescence resonance energy transfer (View Interaction 1, 2)FLS1physically interacts with CHS and DFR by competition binding(View interaction)

Bellis perennis: a useful tool for protein localization studies

Fluorescent fusion proteins together with transient transformation techniques are commonly used to investigate intracellular protein localisation in vivo. Biolistic transfection is reliable, efficient and avoids experimental problems associated with producing and handling fragile protoplasts. Onion epidermis pavement cells are frequently used with this technique, their excellent properties for microscopy resulting from their easy removal from the underlying tissues and large size. They also have advantages over mesophyll cells for fluorescence microscopy, as they are devoid of chloroplasts whose autofluorescence can pose problems. The arrested plastid development is peculiar to epidermal cells, however, and stands in the way of studies on protein targeting to plastids. We have developed a system enabling studies of in vivo protein targeting to organelles including chloroplasts within a photosynthetically active plant cell with excellent optical properties using a transient transformation procedure. We established biolistic transfection in epidermal pavement cells of the lawn daisy (Bellis perennis L., cultivar "Galaxy red") which unusually contain a moderate number of functional chloroplasts. These cells are excellent objects for fluorescence microscopy using current reporters, combining the advantages of the ease of biolistic transfection, the excellent optical properties of a single cell layer and access to chloroplast protein targeting. We demonstrate chloroplast targeting of plastid-localised heme oxygenase, and two further proteins whose localisation was equivocal. We also demonstrate unambiguous targeting to mitochondria, peroxisomes and nuclei. We thus propose that the Bellis system represents a valuable tool for protein localisation studies in living plant cells.

Genomes of obligate plant pathogens reveal adaptations for obligate parasitism

Many destructive crop diseases are caused by filamentous microbes from the kingdoms Fungi (e.g., ascomycetes and basidomycetes) and Stramenopila (oomycetes). Many of these pathogens exploit plants by extracting nutrients from living plant cells (biotrophy) (1). In some cases, biotrophic pathogens have evolved to a state of absolute dependency on their hosts. These obligate biotrophs have lost the capacity for saprotrophic growth, display narrow host specificity, and are difficult or impossible to culture on synthetic media (2). When viewed from an interfacial perspective, these lineages provide fascinating case studies of how microbes can evolve to occupy highly specialized niches inside hostile plant hosts. How is it that these parasites can penetrate and grow within host organs, modify host cell structure, reprogram host metabolism, and reproduce prolifically, all while successfully evading the plant immune system (3)? There is much to be learned about these adaptations from comparative genomics. One such example is provided by a paper in PNAS that describes the genomes of two obligate rust pathogens (basidiomycota) and draws informative comparisons to genomes from related species with different lifestyles (4). Below, I discuss insights from this study in the context of similar analyses of obligate parasites from ascomycetes and oomycetes (5, 6). These comparisons reveal striking commonalities, suggestive of convergent pathways that have been followed as these three lineages independently evolved to an obligate lifestyle.

Auxin Production by the Rice Blast Fungus and its Localization in Host Tissue

AbstractThe rice blast fungus Magnaporthe oryzae requires living plant cells in its early stages of infection and invasion of host tissue. In this study, we showed the possibility that an exogenous fungal auxin could be implicated in hemibiotrophic interactions. Chemical analyses showed that this fungus produces a small amount of the auxin indole‐3‐acetic acid during growth in culture. Immunohistological analyses showed that antigens of the anti‐indole‐3‐acetic acid monoclonal antibody were localized in the fungal infection hyphae present in the rice tissue. Rice transformants containing an auxin‐inducible reporter construct demonstrated an auxin response around the apex of the infection hyphae after fungal invasion of living tissue. These results suggest that the rice blast fungus produces a small amount of auxin during its biotrophic phase and the invaded rice tissue responds to the exogenous fungal auxin. This auxin may be involved in the biotrophic phase of the infection hyphae.

A fast brassinolide‐regulated response pathway in the plasma membrane of Arabidopsis thaliana

To understand molecular processes in living plant cells, quantitative spectro-microscopic technologies are required. By combining fluorescence lifetime spectroscopy with confocal microscopy, we studied the subcellular properties and function of a GFP-tagged variant of the plasma membrane-bound brassinosteroid receptor BRI1 (BRI1-GFP) in living cells of Arabidopsis seedlings. Shortly after adding brassinolide, we observed BRI1-dependent cell-wall expansion, preceding cell elongation. In parallel, the fluorescence lifetime of BRI1-GFP decreased, indicating an alteration in the receptor's physico-chemical environment. The parameter modulating the fluorescence lifetime of BRI1-GFP was found to be BL-induced hyperpolarization of the plasma membrane. Furthermore, for induction of hyperpolarization and cell-wall expansion, activation of the plasma membrane P-ATPase was necessary. This activation required BRI1 kinase activity, and was mediated by BL-modulated interaction of BRI1 with the P-ATPase. Our results were used to develop a model suggesting that there is a fast BL-regulated signal response pathway within the plasma membrane that links BRI1 with P-ATPase for the regulation of cell-wall expansion.

A bright green-colored bimolecular fluorescence complementation assay in living plant cells

Protein-protein interactions are important for various biological and cellular processes. To analyze protein- protein interactions in living cells, the bimolecular fluorescence complementation (BiFC) assay, based on structural complementation of two non-fluorescent N- and C-terminal fragments from a fluorescent protein, has been developed and widely used in various research fields. Here I report a bright green-colored BiFC assay in living plant cells by using the N- terminal fragment (GN) of green fluorescent protein-S65T (GFP-S65T) and the C-terminal fragment (CC) of cyan fluorescent protein (CFP), but not GN and the C-terminal fragment (GC) of GFP. Fluorescence intensity of the GN/CC- based BiFC was 7-fold higher than that of the GN/GC-based BiFC. The emission spectrum of the GN/CC-based BiFC in planta was identical to that of full-length GFP-S65T. Ala163 residue within the CC fragment was found to be responsible for the improvement of the BiFC efficiency. These findings provide a BiFC method for in vivo protein-protein interaction studies to many GFP users in various research fields.