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At least 19 recordsLinked to original sources

Atomic force microscopy of plant cell walls, plant cell wall polysaccharides and gels.

Methods developed for the routine imaging of polysaccharides by atomic force microscopy (AFM) have been used to image plant polysaccharides from higher plants (pectin) and algae (carrageenan). These methods have been extended to image K-carrageenan association in hydrated films. Finally, AFM has been used to image polysaccharide architecture in moist plant cell walls. Simple experimental and image processing methods have been used to enhance molecular structure in 'rough' cell wall surfaces.

Cell Wall↗

Local interactions shape plant cells.

Plant cell expansion is usually attributed to the considerable osmotic pressure that develops within and impinges upon the cell boundary. Whereas turgor containment within expandable walls explains global expansion, the scalar nature of turgor does not directly suggest a mechanism for achieving the localized, differential growth that is responsible for the diversity of plant-cell forms. The key to achieving local growth in plant cells appears to lie not in harnessing turgor but in using it to identify weak regions in the cell boundary and thus creating discrete intracellular domains for targeting the growth machinery. Membrane-interacting phospholipases, Rho-like proteins and their interactors, an actin-modulating ARP2/3 complex with its upstream regulators, and actin-microtubule interactions play important roles in the intracellular cooperation to shape plant cells.

Actin-Related Protein 2-3 Complex↗

Osmoregulation and membrane-mediated responses to altered water potential in plant cells.

Plant cells respond to short-term stress dehydration by modification of internal psi pi such that an inward gradient of psi w is maintained. In response to lowered psi w, increases in internal psi pi are created by alteration of cell inorganic ions and small organic solute content. Passive movement of water follows, changing cell hydration and forcing the plasma membrane against the elastic cell wall. The stretched cell wall presses against the cell contents, creating a hydrostatic pressure, psi p, which tends to force water out of the cell. The resulting hydrostatic pressure eventually comes into equilibrium with forces bringing water into the cell, largely psi pi, and the net flow of water ceases. The mechanism for sensing cell psi w changes is unknown but the initial event must be physical, not biochemical. The method of translation of such physical events into biochemical actions is also unknown but the Zimmermann model provides a means of signal transduction and amplification, through the alteration of membrane parameters, which could account for the observed changes. As for animal cells, cell levels of Ca2+ are important for their regulation of membrane Pj in these responses but unlike osmoregulation in higher animals, the involvement of plant hormones in these responses have not been clearly established. However, the important role of plant cell limiting membranes in plant cell osmoregulation responses seems obvious.

Adaptation, Physiological↗

Divide and conquer: cytokinesis in plant cells.

Plant cells divide in two by constructing a new cell wall (cell plate) between daughter nuclei after mitosis. Golgi-derived vesicles are transported to the equator of a cytoskeletal structure called a phragmoplast, where they fuse together to form the cell plate. Orientation of new cell walls involves actindependent guidance of phragmoplasts and associated cell plates to cortical sites established prior to mitosis. Recent work has provided new insights into how actin filaments and other proteins in the phragmoplast and cell plate contribute to cytokinesis. Newly discovered mutations have identified a variety of genes required for cytokinesis or its spatial regulation.

Cell Division↗

Rapid changes of protein phosphorylation are involved in transduction of the elicitor signal in plant cells.

Plant cells have an acute sense for pathogen-derived chemical stimuli, so-called elicitors, which induce the plant's defense response. To investigate the molecular basis of chemosensory transduction, elicitor-treated tomato cells were labeled with 1-min pulses of [32P] phosphate. This technique revealed drastic changes in protein phosphorylation in vivo within minutes of stimulation. The protein kinase inhibitors K-252a and staurosporine completely prevented these elicitor-induced changes in protein phosphorylation. They also blocked two early biochemical responses to elicitors, extracellular alkalinization and biosynthesis of ethylene. The ability of K-252a, staurosporine, and benzoylated staurosporine derivatives to inhibit elicitor responses in vivo correlated with their ability to inhibit tomato microsomal protein kinase in vitro. When K-252a was given to elicited cells 1 min after the[32] phosphate, the radioactivity in certain newly labeled phosphoprotein bands disappeared again within minutes. This correlated with an arrest of alkalinization within minutes when K-252a was applied in midcourse of elicitation. These data show that phosphorylation of protein substrates by K-252a-sensitive protein kinases is essential for transduction of elicitor signals in plant cells and that continuous phosphorylation of these proteins is required to maintain the elicited state.

Journal Article↗

Detection of in vivo protein interactions between Snf1-related kinase subunits with intron-tagged epitope-labelling in plants cells.

Plant orthologs of the yeast sucrose non-fermenting (Snf1) kinase and mammalian AMP-activated protein kinase (AMPK) represent an emerging class of important regulators of metabolic and stress signalling. The catalytic alpha-subunits of plant Snf1-related kinases (SnRKs) interact in the yeast two-hybrid system with different proteins that share conserved domains with the beta- and gamma-subunits of Snf1 and AMPKs. However, due to the lack of a robust technique allowing the detection of protein interactions in plant cells, it is unknown whether these proteins indeed occur in SnRK complexes in vivo. Here we describe a double-labelling technique, using intron-tagged hemagglutinin (HA) and c-Myc epitope sequences, which provides a simple tool for co-immunopurification of interacting proteins expressed in Agrobacterium-transformed Arabidopsis cells. This generally applicable plant protein interaction assay was used to demonstrate that AKINbeta2, a plant ortholog of conserved Snf1/AMPK beta-subunits, forms different complexes with the catalytic alpha-subunits of Arabidopsis SnRK protein kinases AKIN10 and AKIN11 in vivo.

Arabidopsis↗

Induction of defense responses in cultured parsley cells by plant cell wall fragments.

Cell suspension cultures of parsley (Petroselinum crispum) accumulated coumarin phytoalexins and exhibited increased beta-1,3-glucanase activity when treated with either a purified alpha-1,4-d-endopolygalacturonic acid lyase from Erwinia carotovora or oligogalacturonides solubilized from parsley cell walls by endopolygalacturonic acid lyase. Coumarin accumulation induced by the plant cell wall elicitor was preceded by increases in the activities of phenylalanine ammonia lyase (PAL), 4-coumarate:CoA ligase (4CL) and S-adenosyl-l-methionine:xanthotoxol O-methyltransferase (XMT). The time courses for the changes in these three enzyme activities were similar to those observed in cell cultures treated with a fungal glucan elicitor. The plant cell wall elicitor was found to act synergistically with the fungal glucan elicitor in the induction of coumarin phytoalexins. As much as a 10-fold stimulation in coumarin accumulation above the calculated additive response was observed in cell cultures treated with combinations of plant and fungal elicitors. The synergistic effect was also observed for the induction of PAL, 4CL, and XMT activities. These results demonstrate that plant cell wall elicitors induce at least two distinct biochemical responses in parsley cells and further support the role of oligogalacturonides as important regulators of plant defense.

Journal Article↗

Shaping in plant cells.

Plant cells adopt a diversity of different shapes that are adapted to their specific functions. Central to the development of specialised form is the modification of cell-wall composition and organisation. A number of recent papers emphasise the importance of the cell wall to cell shaping, in the definition of both localised regions that are expandable and regions that are more resistant to mechanical forces. The organisation and activity of the cytoskeleton, and the activity of signalling pathways, are also essential in defining regions of the cell wall that will grow and those that will not. Although turgor has long been assumed to be a rather passive contributor to cell shaping, recent reports show that, in some cells, differential changes in turgor may have a role in establishing specialised cell form.

Cell Size↗

Production of a foreign protein product with genetically modified plant cells.

Plant cells (Nicotiana tabacum) were genetically engineered to produce a foreign protein, chloramphenicol acetyltransferase (CAT), and the CAT production from suspension cultures was investigated. Suspension cultures were grown in a shake flask, a stirred fermenter, and a bubble-column fermenter. The CAT production was growth related and the maximum activity was reached during the early stationary phase. A 41-day, semicontinuous stirred fermenter run, consisting of five sequential batch runs, demonstrated long-term CAT production. Continuous CAT production was also accomplished in a bubble-column fermenter at a medium flow rate of 3.1 ml h-1, which was equivalent to a dilution rate of 0.25 day-1.

Cells, Cultured↗

Potassium homeostasis in vacuolate plant cells.

Plant cells contain two major pools of K+, one in the vacuole and one in the cytosol. The behavior of K+ concentrations in these pools is fundamental to understanding the way this nutrient affects plant growth. Triple-barreled microelectrodes have been used to obtain the first fully quantitative measurements of the changes in K+ activity (aK) in the vacuole and cytosol of barley (Hordeum vulgare L.) root cells grown in different K+ concentrations. The electrodes incorporate a pH-selective barrel allowing each measurement to be assigned to either the cytosol or vacuole. The measurements revealed that vacuolar aK declined linearly with decreases in tissue K+ concentration, whereas cytosolic aK initially remained constant in both epidermal and cortical cells but then declined at different rates in each cell type. An unexpected finding was that cytoplasmic pH declined in parallel with cytosolic aK, but acidification of the cytosol with butyrate did not reveal any short-term link between these two parameters. These measurements show the very different responses of the vacuolar and cytosolic K+ pools to changes in K+ availability and also show that cytosolic K+ homeostasis differs quantitatively in different cell types. The data have been used in thermodynamic calculations to predict the need for, and likely mechanisms of, active K+ transport into the vacuole and cytosol. The direction of active K+ transport at the vacuolar membrane changes with tissue K+ status.

Journal Article↗

Elimination of transcriptional interference between tandem genes in plant cells.

Plant cells are commonly transformed with two or more tandemly arranged genes, but how orientation affects their expression is not well understood. We investigated the amount of transcriptional interference occurring between two adjacent genes by cloning luciferase and green fluorescent protein (GFP) genes (promoter--coding sequence--terminator) in all possible orientations and expressing the genes in tobacco protoplasts. When two genes are oriented head-to-tail (-->-->), the expression of the downstream gene was reduced 80% by the upstream gene. When two genes are oriented tail-to-tail (--><--), the expression of the upstream gene was reduced 53% by the expression of the downstream gene. There was no interference when the orientation was head-to-head (<---->). Using a chemically inducible gene expression system, we showed that the downstream gene expression was reduced 71% by the induction of an upstream gene. Inserting a mammalian transcription blocker sequence eliminated the interference between the genes in tail-to-tail orientation. The interference in the head-to-tail orientation was eliminated by inserting a 2322-bp lambda phage DNA fragment. The terminators in gene constructs did not prevent the transcriptional interference, and the interference was eliminated by designing the orientation of genes and by placing a transcription blocker or a lambda phage sequence between genes.

Bacteriophage lambda↗

Large-scale micropropagation system of plant cells.

Plant micropropagation is an efficient method of propagating disease-free, genetically uniform and massive amounts of plants in vitro. The scale-up of the whole process for plant micropropagation should be established by an economically feasible technology for large-scale production of them in appropriate bioreactors. It is necessary to design suitable bioreactor configuration which can provide adequate mixing and mass transfer while minimizing the intensity of shear stress and hydrodynamic pressure. Automatic selection of embryogenic calli and regenerated plantlets using image analysis system should be associated with the system. The aim of this chapter is to identify the problems related to large-scale plant micropropagation via somatic embryogenesis, and to summarize the micropropagation technology and computer-aided image analysis. Viscous additive supplemented culture, which is including the successful results obtained by us for callus regeneration, is also introduced.

Alginates↗

Drought-induced responses in plant cells.

Plants subjected to water stress undergo numerous physiological and metabolic changes. A general decrease in photosynthetic rate is among the most common responses. This is due to a programmed process involving the closure of stomata and reduction in the activity of photosynthetic enzymes. The plant hormone abscisic acid plays an important role in this process. Accumulation of compatible solutes, during water stress, is thought to be an adaptive response which has been developed by some plant species. Engineering the genes involved in the synthesis of these compounds, into nonaccumulating plants, has demonstrated promising results for genetic improvement of drought tolerance. Drought stress induces alteration of gene expression. A large number of genes which are upregulated by water stress have been isolated and characterized. Proteins encoded by some of these genes share several characteristics. The biochemical role of most of these gene products is unknown, but potential adaptive functions have been suggested. Abscisic acid is involved in the regulation of some of these genes.

Adaptation, Physiological↗

Molecular recognition of pathogen attack occurs inside of plant cells in plant disease resistance specified by the Arabidopsis genes RPS2 and RPM1.

The Arabidopsis thaliana disease resistance genes RPS2 and RPM1 belong to a class of plant disease resistance genes that encode proteins that contain an N-terminal tripartite nucleotide binding site (NBS) and a C-terminal tandem array of leucine-rich repeats. RPS2 and RPM1 confer resistance to strains of the bacterial phytopathogen Pseudomonas syringae carrying the avirulence genes avrRpt2 and avrB, respectively. In these gene-for-gene relationships, it has been proposed that pathogen avirulence genes generate specific ligands that are recognized by cognate receptors encoded by the corresponding plant resistance genes. To test this hypothesis, it is crucial to know the site of the potential molecular recognition. Mutational analysis of RPS2 protein and in vitro translation/translocation studies indicated that RPS2 protein is localized in the plant cytoplasm. To determine whether avirulence gene products themselves are the ligands for resistance proteins, we expressed the avrRpt2 and avrB genes directly in plant cell using a novel quantitative transient expression assay, and found that expression of avrRpt2 and avrB elicited a resistance response in plants carrying the corresponding resistance genes. This observation indicates that no bacterial factors other than the avirulence gene products are required for the specific resistance response as long as the avirulence gene products are correctly localized. We propose that molecular recognition of P. syringae in RPS2- and RPM1-specified resistance occurs inside of plant cells.

Amino Acid Sequence↗

Spatial aspects of cytokinesis in plant cells.

Plant cytokinesis in a particular orientation and location can be viewed as having several component stages, often beginning with the establishment of division polarity before karyokinesis occurs. Improved methods for preserving the in situ distribution of actin microfilaments and observations of individual live cells during treatment with cytoskeleton-disrupting drugs are making it possible to elucidate the roles of microtubules and microfilaments in cytokinesis. Current evidence points to involvement of the cytoskeleton throughout the stages of preparation for, and execution of, cytokinesis in many types of plant cell division.

Actins↗

New aspects of gravity responses in plant cells.

Plants show two distinct responses to gravity: gravity-dependent morphogenesis (gravimorphogenesis) and gravity resistance. In gravitropism, a typical mechanism of gravimorphogenesis, gravity is utilized as a signal to establish an appropriate form. The response has been studied in a gravity-free environment, where plant seedlings were found to perform spontaneous morphogenesis, termed automorphogenesis. Automorphogenesis consists of a change in growth direction and spontaneous curvature in dorsiventral directions. The spontaneous curvature is caused by a difference in the capacity of the cell wall to expand between the dorsal and the ventral sides of organs, which originates from the inherent structural anisotropy. Gravity resistance is a response that enables the plant to develop against the gravitational force. To resist the force, the plant constructs a tough body by increasing the cell wall rigidity that suppresses growth. The mechanical properties of the cell wall are changed by modification of the cell wall metabolism and cell wall environment, especially pH. In gravitropism, gravity is perceived by amyloplasts in statocytes, whereas gravity resistance may be mediated by mechanoreceptors on the plasma membrane.

Cell Wall↗

Constitutive caspase-like machinery executes programmed cell death in plant cells.

The morphological features of programmed cell death (PCD) and the molecular machinery involved in the death program in animal cells have been intensively studied. In plants, cell death has been widely observed in predictable patterns throughout differentiation processes and in defense responses. Several lines of evidence argue that plant PCD shares some characteristic features with animal PCD. However, the molecular components of the plant PCD machinery remain obscure. We have shown that plant cells undergo PCD by constitutively expressed molecular machinery upon induction with the fungal elicitor EIX or by staurosporine in the presence of cycloheximide. The permeable peptide caspase inhibitors, zVAD-fmk and zBocD-fmk, blocked PCD induced by EIX or staurosporine. Using labeled VAD-fmk, active caspase-like proteases were detected within intact cells and in cell extracts of the PCD-induced cells. These findings suggest that caspase-like proteases are responsible for the execution of PCD in plant cells.

Amino Acid Chloromethyl Ketones↗