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In this study, we identified and functionally characterized the mitochondrial heat shock protein 70 (mtHsp70). Over-expression of mtHsp70 suppressed heat- and H(2)O(2)-induced programmed cell death (PCD) in rice protoplasts, as reflected by higher cell viability, decreased DNA laddering and chromatin condensation. Mitochondrial membrane potential (Δψ(m)) after heat shock was destroyed gradually in protoplasts, but mtHsp70 over-expression showed higher Δψ(m) relative to the vector control cells, and partially inhibited cytochrome c release from mitochondria to cytosol. Heat treatment also significantly increased reactive oxygen species (ROS) generation, a phenomenon not observed in protoplasts over-expressing mtHsp70. Together, these results suggest that mtHsp70 may suppress PCD in rice protoplasts by maintaining mitochondrial Δψ(m) and inhibiting the amplification of ROS., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Regulation of gene expression by transcription factors is a fundamental mechanism in essentially all aspects of cellular processes. Transient expression assay of a reporter plasmid containing a reporter gene driven by a promoter of interest and an effector plasmid expressing a transcription factor has been a powerful tool for analyzing transcription factors. Here we present a protocol for polyethylene glycol (PEG)-mediated transformation of Arabidopsis protoplasts. It details preparation of protoplasts from Arabidopsis suspension cultured cells or leaves of soil-grown Arabidopsis plants and subsequent PEG-mediated transformation with reporter and effector plasmids. This protocol can be completed within 24 h from protoplast preparation to reporter assay. As an example, analysis of the membrane-bound transcription factor AtbZIP60 and its target BiP3 promoter is shown.

Methods are described which allow one to observe chloroplasts in mesophyll cells from leaves of Arabidopsis, determine their number per cell, measure their area, and determine a value for chloroplast coverage inside mesophyll cells. Non-green plastids can also be imaged either by using staining, or by exploiting fluorescent proteins targeted to the plastid in non-green parts of the plant, such as the roots, in transgenic Arabidopsis.

There is increasing concern regarding the use of fungicides to control plant diseases, whereby interest has increased in the biological control of phytopathogenic fungi by the application of hypovirulent mycoviruses as a possible alternative to fungicides. Transmission of hypovirulence-associated double-stranded RNA (dsRNA) viruses between mycelia, however, is prevented by the vegetative incompatibility barrier that often exists between different species or strains of filamentous fungi. We determined whether protoplast fusion could be used to transmit FgV1-DK21 virus, which is associated with hypovirulence on F. boothii (formerly F. graminearum strain DK21), to F. graminearum, F. asiaticum, F. oxysporum f. sp. lycopersici, and Cryphonectria parasitica. Relative to virus-free strains, the FgV1-DK21 recipient strains had reduced growth rates, altered pigmentation, and reduced virulence. These results indicate that protoplast fusion can be used to introduce FgV1-DK21 dsRNA into other Fusarium species and into C. parasitica and that FgV1-DK21 can be used as a hypovirulence factor and thus as a biological control agent.

Transplastomic tobacco (Nicotiana tabacum) plants expressing β-glucosidase (Bgl-1) show modified development. They flower 1 month earlier with an increase in biomass (1.9-fold), height (1.5-fold), and leaf area (1.6-fold) than untransformed plants. Trichome density on the upper and lower leaf surfaces of BGL-1 plants increase by 10- and 7-fold, respectively, harboring 5-fold more glandular trichomes (as determined by rhodamine B staining), suggesting that BGL-1 lines produce more sugar esters than control plants. Gibberellin (GA) levels were investigated because it is a known regulator of flowering time, plant height, and trichome development. Both GA(1) and GA(4) levels are 2-fold higher in BGL-1 leaves than in untransformed plants but do not increase in other organs. In addition, elevated levels of other plant hormones, including zeatin and indole-3-acetic acid, are observed in BGL-1 lines. Protoplasts from BGL-1 lines divide and form calli without exogenous hormones. Cell division in protoplasts is enhanced 7-fold in the presence of exogenously applied zeatin-O-glucoside conjugate, indicating the release of active hormones from their conjugates. Whitefly (Bemisia tabaci) and aphid (Myzus persicae) populations in control plants are 18 and 15 times higher than in transplastomic lines, respectively. Lethal dose to kill 50% of the test population values of 26.3 and 39.2 μg per whitefly and 23.1 and 35.2 μg per aphid for BGL-1 and untransformed control exudates, respectively, confirm the enhanced toxicity of transplastomic exudates. These data indicate that increase in sugar ester levels in BGL-1 lines might function as an effective biopesticide. This study provides a novel strategy for designing plants for enhanced biomass production and insect control by releasing plant hormones or sugar esters from their conjugates stored within their chloroplasts.

In plant cells, phospholipids are not only membrane components but also act as second messengers interacting with various proteins and regulating diverse cellular processes, including stress signal transduction. Here, we report studies on the effects of various phospholipids on the activity and expression of maize wound-responsive calcium-dependent protein kinase (ZmCPK11). Our results revealed that in leaves treated with n-butanol, a potent inhibitor of phosphatidic acid (PA) synthesis catalyzed by phospholipase D, a significant decrease of ZmCPK11 activity was observed, indicating contribution of PA in the kinase activation. Using lipid binding assays, we demonstrate that among various phospholipids only saturated acyl species (16:0 and 18:0) of phosphatidic acid are able to bind to ZmCPK11. Saturated acyl species of PA are also able to stimulate phosphorylation of exogenous substrates by ZmCPK11 and autophosphorylation of the kinase. The level of ZmCPK11 autophosphorylation is correlated with its enzymatic activity. RT-PCR analysis showed that transcript level of ZmCPK11 in maize leaves increased in response to PA treatment. The influence of PA on the activity and transcript level of ZmCPK11 suggests an involvement of this kinase in a PA-mediated wound signal transduction pathway.

The cyclic nucleotide cGMP has been shown to play important roles in plant development and responses to abiotic and biotic stress. To date, the techniques that are available to measure cGMP in plants are limited by low spatial and temporal resolution. In addition, tissue destruction is necessary. To circumvent these drawbacks we have used the δ-FlincG fluorescent protein to create an endogenous cGMP sensor that can report cellular cGMP levels with high resolution in time and space in living plant cells. δ-FlincG in transient and stably expressing cells shows a dissociation constant for cGMP of around 200 nm giving it a dynamic range of around 20-2000 nm. Stimuli that were previously shown to alter cGMP in plant cells (nitric oxide and gibberrellic acid) evoked pronounced fluorescence signals in single cells and in root tissues, providing evidence that δ-FlincG reports changes in cellular cGMP in a physiologically relevant context., (© 2010 The Authors. The Plant Journal © 2010 Blackwell Publishing Ltd.)

Arabidopsis thaliana AtNUDT7 Nudix pyrophosphatase hydrolyzes NADH and ADP-ribose in vitro and is an important factor in the cellular response to diverse biotic and abiotic stresses. Several studies have shown that loss-of-function Atnudt7 mutant plants display many profound phenotypes. However the molecular mechanism of AtNUDT7 function remains elusive. To gain a better understanding of this hydrolase cellular role, proteins interacting with AtNUDT7 were identified. Using AtNUDT7 as a bait in an in vitro binding assay of proteins derived from cultured Arabidopsis cell extracts we identified the regulatory protein RACK1A as an AtNUDT7-interactor. RACK1A-AtNUDT7 interaction was confirmed in a yeast two-hybrid assay and in a pull-down assay and in Bimolecular Fluorescence Complementation (BiFC) analysis of the proteins transiently expressed in Arabidopsis protoplasts. However, no influence of RACK1A on AtNUDT7 hydrolase catalytic activity was observed. In vitro interaction between RACK1A and the AGG1 and AGG2 gamma subunits of the signal transducing heterotrimeric G protein was also detected and confirmed in BiFC assays. Moreover, association between AtNUDT7 and both AGG1 and AGG2 subunits was observed in Arabidopsis protoplasts, although binding of these proteins could not be detected in vitro. Based on the observed interactions we conclude that the AtNUDT7 Nudix hydrolase forms complexes in vitro and in vivo with regulatory proteins involved in signal transduction. Moreover, we provide the initial evidence that both signal transducing gamma subunits bind the regulatory RACK1A protein.

Protein-protein interactions are an important aspect of the gene regulation process. The expression of a gene in response to certain stimuli, within a specific cell type or at a particular developmental stage, involves a complex network of interactions between different regulatory proteins and the cis-regulatory elements present in the promoter of the gene. A number of methods have been developed to study protein-protein interactions in vitro and in vivo in plant cells, one of which is bimolecular fluorescence complementation (BiFC). BiFC is a relatively simple technique based upon the reconstitution of a fluorescent protein. The interacting protein complex can be visualized directly in a living plant cell when two non-fluorescent fragments, of an otherwise fluorescent protein, are fused to proteins found within that complex. Interaction of tagged proteins brings the two non-fluorescent fragments into close proximity and reconstitutes the fluorescent protein. In addition, the subcellular location of an interacting protein complex in the cell can be simultaneously determined. Using this approach, we have successfully demonstrated a protein-protein interaction between a R2R3 MYB and a basic helix-loop-helix MYC transcription factor related to flavonoid biosynthetic pathway in tobacco protoplasts.

Background: Post transcriptional gene silencing (PTGS) is a mechanism harnessed by plant biologists to knock down gene expression. siRNAs contribute to PTGS that are synthesized from mRNAs or viral RNAs and function to guide cellular endoribonucleases to target mRNAs for degradation. Plant biologists have employed electroporation to deliver artificial siRNAs to plant protoplasts to study gene expression mechanisms at the single cell level. One drawback of electroporation is the extensive loss of viable protoplasts that occurs as a result of the transfection technology., Results: We employed fluorescent conjugated polymer nanoparticles (CPNs) to deliver siRNAs and knockdown a target gene in plant protoplasts. CPNs are non toxic to protoplasts, having little impact on viability over a 72 h period. Microscopy and flow cytometry reveal that CPNs can penetrate protoplasts within 2 h of delivery. Cellular uptake of CPNs/siRNA complexes were easily monitored using epifluorescence microscopy. We also demonstrate that CPNs can deliver siRNAs targeting specific genes in the cellulose biosynthesis pathway (NtCesA-1a and NtCesA-1b)., Conclusions: While prior work showed that NtCesA-1 is a factor involved in cell wall synthesis in whole plants, we demonstrate that the same gene plays an essential role in cell wall regeneration in isolated protoplasts. Cell wall biosynthesis is central to cell elongation, plant growth and development. The experiments presented here shows that NtCesA is also a factor in cell viability. We show that CPNs are valuable vehicles for delivering siRNAs to plant protoplasts to study vital cellular pathways at the single cell level.

Electroporation--the use of high-voltage electric shocks to introduce DNA into cells--can be used with most cell types, yields a high frequency of both stable transformation and transient gene expression, and, because it requires fewer steps, can be easier than alternate techniques. This unit describes electroporation of mammalian cells, including ES cells for the preparation of knock-out, knock-in, and transgenic mice. Protocols are described for the use of electroporation in vivo to perform gene therapy for cancer therapy and DNA vaccination. Also described are modifications for preparation and transfection of plant protoplasts., (© 2010 by John Wiley & Sons, Inc.)

Two different endophytic strains, ESRAA1997 and ALAA2000, were isolated from the Egyptian herbal plant Anastatica hierochuntica. The 2 strains produced alkaline serine protease and were identified based on their phenotypic and chemotypic characteristics as different strains of Micromonospora spp. Both strains grew and produced keratinase, using different keratinous waste substances as the sole source of carbon and nitrogen. In our study, the activity and properties of keratinase enzymes of the wild strains ESRAA1997 and ALAA2000 were altered by genetic recombination through protoplast fusion between them, leading to a potent keratinolytic fusant Micromonospora strain AYA2000 with improved properties (activity, stability, specificity, and tolerance to inhibitors). Using a mixture of yeast extract, peptone, and malt extract as a supplement to the bovine hair medium increased keratinase production by 48%, and addition of 1% glucose suppressed enzyme production by Micromonospora strain AYA2000. The enzyme was purified by ammonium sulphate precipitation and DEAE-cellulose chromatography followed by gel filtration. The molecular weight, estimated using SDS-PAGE, was 39 kDa. The enzyme exhibited remarkable activity towards all keratinous wastes used and could also adapt to a broad range of pH and temperatures, with optima at pH 11 and 60 °C. The enzyme was not influenced by chelating reagents, metal ions, or alcohols. These properties make AYA2000 keratinase an ideal candidate for biotechnological application.

RNA-induced silencing complexes (RISCs) play central roles in posttranscriptional gene silencing. In plants, the mechanism of RISC assembly has remained elusive due to the lack of cell-free systems that recapitulate the process. In this report, we demonstrate that plant AGO1 protein synthesized by in vitro translation using an extract of evacuolated tobacco protoplasts incorporates synthetic small interfering RNA (siRNA) and microRNA (miRNA) duplexes to form RISCs that sequester the single-stranded siRNA guide strand and miRNA strand, respectively. The formed RISCs were able to recognize and cleave the complementary target RNAs. In this system, the siRNA duplex was incorporated into HSP90-bound AGO1, and subsequent removal of the passenger strand was triggered by ATP hydrolysis by HSP90. Removal of the siRNA passenger strand required the ribonuclease activity of AGO1, while that of the miRNA star strand did not. Based on these results, the mechanism of plant RISC formation is discussed., (Copyright 2010 Elsevier Inc. All rights reserved.)

A methodology is described for the quantitative determination of Förster resonance energy transfer (FRET) in live cells using the rise time of acceptor fluorescence as determined with fluorescence lifetime imaging microscopy (FLIM). An advantage of this method is that only those molecules that are involved in the energy-transfer process are monitored. This contrasts with current methods that measure either steady-state fluorescence of donor and acceptor molecules or time-resolved fluorescence of donor molecules, and thereby probe a mixture of donor molecules that are involved in FRET and those that are fluorescent but not involved in FRET. The absence of FRET can, for instance, be due to unwanted acceptor bleaching or incomplete maturing of visible proteins that should act as acceptor molecules. In addition, parameters describing the rise of acceptor fluorescence and the decay of donor fluorescence can be determined via simultaneous global analysis of multiple FLIM images, thereby increasing the reliability of the analysis. In the present study, plant protoplasts transfected with fusions of visible fluorescent proteins are used to illustrate the new data analysis method. It is demonstrated that the distances estimated with the present method are substantially smaller than those estimated from the average donor lifetimes, due to a fraction of non-transferring donor molecules. Software to reproduce the presented results is provided in an open-source and freely available package called "TIMP" for "The R project for Statistical Computing".

(19)F NMR is a unique tool to examine the structure of fluorine-labeled peptides in their native cellular environment, due to an exquisite sensitivity and lack of natural abundance background. For solid-state NMR analysis, we isolated native membranes from erythrocyte ghosts and bacterial protoplasts and prepared them as macroscopically oriented samples. They showed a high purity and quality of alignment according to (31)P NMR, and the membrane-bound antimicrobial peptide PGLa could be detected by (19)F NMR. The characteristic fingerprint splitting of its (19)F reporter group indicated that the peptide helix binds to the native membranes in a surface alignment, albeit with a higher affinity in the prokaryotic than the eukaryotic system.

Objective: To acquire homozygous tetraploid germplasm of Rhodiola sachalinensis., Method: PEG-mediated protoplast fusions were conducted using callus of Rh. sachalinensis as materials. Protoplast fusion products were embedded and cultured in low-density, low-melting-point agar and marked according to the protoplast size, and single-celled sister lines were established to acquire genetically homozygous tetraploid germplasm., Result: R(D) and R(M) of newborn daughter cells or protoplasm, metaphase cells or protoplasm were approximately in line with the formula R(D) = 0.793 7R(M). The change range in diameter of the diploid cells without fusion, two protoplasts fusion product were: 16.7 microm < or = R < 21.3 microm, 21.0 microm < or = R' < 26.8 microm respectively. There is an overlap between the two diameter ranges. The protoplast inoculation density of 1 x 10(4) cells x mL(-1) was appropriate when protoplasts were anchored by low-intensity, low-melting-point agar. Under the conditions of this density, plating efficiency was high and single cell origin of the sister lines microclones grew rapidly, and it was easy to mark the single cell microclones, and separate from each other to subculture. The chromosome counts results showed that chromosome numbers of diploid and tetraploid of single cell lines were 26 and 52, respectively. The result from flow cytometry assay showed that there is no presence of chimerism in single-cell regeneration plantlets., Conclusion: The results of this study provide a scientific basis for polyploid breeding of Rh. sachalinensis.

Using confocal microscopy, X-ray microanalysis and the scanning ion-selective electrode technique, we investigated the signalling of H(2)O(2), cytosolic Ca(2+) ([Ca(2+)](cyt)) and the PM H(+)-coupled transport system in K(+)/Na(+) homeostasis control in NaCl-stressed calluses of Populus euphratica. An obvious Na(+)/H(+) antiport was seen in salinized cells; however, NaCl stress caused a net K(+) efflux, because of the salt-induced membrane depolarization. H(2)O(2) levels, regulated upwards by salinity, contributed to ionic homeostasis, because H(2)O(2) restrictions by DPI or DMTU caused enhanced K(+) efflux and decreased Na(+)/H(+) antiport activity. NaCl induced a net Ca(2+) influx and a subsequent rise of [Ca(2+)](cyt), which is involved in H(2)O(2)-mediated K(+)/Na(+) homeostasis in salinized P. euphratica cells. When callus cells were pretreated with inhibitors of the Na(+)/H(+) antiport system, the NaCl-induced elevation of H(2)O(2) and [Ca(2+)](cyt) was correspondingly restricted, leading to a greater K(+) efflux and a more pronounced reduction in Na(+)/H(+) antiport activity. Results suggest that the PM H(+)-coupled transport system mediates H(+) translocation and triggers the stress signalling of H(2)O(2) and Ca(2+), which results in a K(+)/Na(+) homeostasis via mediations of K(+) channels and the Na(+)/H(+) antiport system in the PM of NaCl-stressed cells. Accordingly, a salt stress signalling pathway of P. euphratica cells is proposed.

As single cell systems, protoplasts have been used in physiological, biochemical and molecular studies aiming towards the investigation, improvement or modification of plants. In grapevine, protoplasts have been isolated from several source tissues but not from grape berry, a major challenge given the uniqueness of grape fruit for human diet and wine production. Also, as the ripe grape berry has long been considered a 'small bag of sugary water' without cell compartmentation and/or membrane integrity, the isolation of intact cells from the mesocarp is of special scientific significance. Protoplasting from grape berry mesocarp cells was achieved with cellulase and pectolyase digestion, followed by differential and gradient centrifugations; however, given the special characteristics of berry tissue, cell wall digestion and protoplast purification were performed in a special environment to maintain their integrity and viability. Light and epifluorescence microscopy revealed the spatial organization of the cytoplasm, where an intricate acidic vacuolar apparatus predominates supporting the idea that berry softening during ripening is not strictly associated with loss in compartmentation and/or membrane integrity. Following the worldwide economical and social importance of wine in modern days, grape berry protoplasts are a major advance for both basic research of fruit ripening and biotechnological applications.

A water-soluble immunoenhancing polysaccharide was isolated from the aqueous extract of fruit bodies of somatic hybrid (Pflo Vv5 FB), obtained through protoplast fusion between Pleurotus florida and Volvariella volvacea strains. On the basis of acid hydrolysis, the polysaccharide was found to contain glucose only. Methylation analysis, periodate oxidation along with (1)H, DEPT-135, and (13)C NMR spectroscopy, including two-dimensional TOCSY, DQF-COSY, NOESY, ROESY, 1H,13C-HMQC, and HMBC experiments showed that the polysaccharide was a (1-->6)-beta-d-glucan, which was not a constituent of any of the parent mushrooms previously reported. This glucan stimulated the macrophages, splenocytes, and thymocytes.

Reactive oxygen species (ROS) are central to plant stress response, signalling, development and a multitude of other processes. In this study, the plasma-membrane hydroxyl radical (HR)-activated K(+) channel responsible for K(+) efflux from root cells during stress accompanied by ROS generation is characterised. The channel showed 16-pS unitary conductance and was sensitive to Ca(2+), tetraethylammonium, Ba(2+), Cs(+) and free-radical scavengers. The channel was not found in the gork1-1 mutant, which lacks a major plasma-membrane outwardly rectifying K(+) channel. In intact Arabidopsis roots, both HRs and stress induced a dramatic K(+) efflux that was much smaller in gork1-1 plants. Tests with electron paramagnetic resonance spectroscopy showed that NaCl can stimulate HR generation in roots and this might lead to K(+)-channel activation. In animals, activation of K(+)-efflux channels by HRs can trigger programmed cell death (PCD). PCD symptoms in Arabidopsis roots developed much more slowly in gork1-1 and wild-type plants treated with K(+)-channel blockers or HR scavengers. Therefore, similar to animal counterparts, plant HR-activated K(+) channels are also involved in PCD. Overall, this study provides new insight into the regulation of plant cation transport by ROS and demonstrates possible physiological properties of plant HR-activated K(+) channels.

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis. An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: P(SCR;)::GFP (endodermis and quiescent center) and P(WOX5;)::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution. FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time. This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

Although there is evident homology among reproductive organs when comparing Poaceae (grass) and eudicots, the identity of grass specific organs, such as lodicules, palea, lemma, and glumes has been the subject of a vast and largely inconclusive discussion. Here we provide some direct evidence to support the idea that the empty glumes of rice (Oryza sativa) are counterparts of lemmas. We show that the development of empty glumes is regulated by ELE (elongated empty glume), which belongs to a plant specific novel gene family. Mutations at the ELE locus cause elongated empty glumes, which mimic the lemmas and have the epidermal morphology of lemmas with four or five vascular bundles. As a nuclear-localized gene, ELE is specifically expressed at the empty glumes of immature spikelets, and its ectopic expression causes many floral development defects, including lemma-like palea, extra palea-like structures, elongated lodicules, extra stamens and stigmas. Our result suggests that empty glumes are lemmas of the sterile florets located at the lateral side of the rice spikelet, and ELE acts as a regulator restraining its growth to maintain its small size in wild-type plants., (Copyright 2010 Institute of Genetics and Developmental Biology and the Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

The present study suggests the importance of reactive oxygen species (ROS) and antioxidant metabolites as biochemical signals during the beneficial interactions of mitochondrial metabolism with photosynthetic carbon assimilation at saturating light and optimal CO2. Changes in steady-state photosynthesis of pea mesophyll protoplasts monitored in the presence of antimycin A [AA, inhibitor of cytochrome oxidase (COX) pathway] and salicylhydroxamic acid [SHAM, inhibitor of alternative oxidase (AOX) pathway] were correlated with total cellular ROS and its scavenging system. Along with superoxide dismutase (SOD) and catalase (CAT), responses of enzymatic components--ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), glutathione reductase (GR) and non-enzymatic redox components of ascorbate-glutathione (Asc-GSH) cycle, which play a significant role in scavenging cellular ROS, were examined in the presence of mitochondrial inhibitors. Both AA and SHAM caused marked reduction in photosynthetic carbon assimilation with concomitant rise in total cellular ROS. Restriction of electron transport through COX or AOX pathway had differential effect on ROS generating (SOD), ROS scavenging (CAT and APX) and antioxidant (Asc and GSH) regenerating (MDAR and GR) enzymes. Further, restriction of mitochondrial electron transport decreased redox ratios of both Asc and GSH. However, while decrease in redox ratio of Asc was more prominent in the presence of SHAM in light compared with dark, decrease in redox ratio of GSH was similar in both dark and light. These results suggest that the maintenance of cellular ROS at optimal levels is a prerequisite to sustain high photosynthetic rates which in turn is regulated by respiratory capacities of COX and AOX pathways.

Regulation of gene expression is largely coordinated by a complex network of interactions between transcription factors (TFs), co-factors, and their cognate cis-regulatory elements in the genome. TFs are multidomain proteins that arise evolutionarily through protein domain shuffling. The modular nature of TFs has led to the idea that specific modules of TFs can be re-designed to regulate desired gene(s) through protein engineering. Utilization of designer TFs for the control of metabolic pathways has emerged as an effective approach for metabolic engineering. We are interested in engineering the basic helix-loop-helix (bHLH, Myc-type) transcription factors. Using site-directed and saturation mutagenesis, in combination with efficient and high-throughput screening systems, we have identified and characterized several amino acid residues critical for higher transactivation activity of a Myc-like bHLH transcription factor involved in anthocyanin biosynthetic pathway in plants. Site-directed and saturation mutagenesis should be generally applicable to engineering of all TFs.

In Arabidopsis, CORYNE (CRN), a new member of the receptor kinase family, was recently isolated as a key player involved in the CLAVATA3 (CLV3) signaling pathway, thereby playing an important role in regulating the development of shoot and root apical meristems. However, the precise relationships among CLAVATA1 (CLV1), CLAVATA2 (CLV2), and CRN receptors remain unclear. Here, we demonstrate the subcellular localization of CRN and analyze the interactions among CLV1, CLV2, and CRN using firefly luciferase complementation imaging (LCI) assays in both Arabidopsis mesophyll protoplasts and Nicotiana benthamiana leaves. Fluorescence targeting showed that CRN was localized to the plasma membrane. The LCI assays coupled with co-immunoprecipitation assays demonstrated that CLV2 can directly interact with CRN in the absence of CLV3. Additional LCI assays showed that CLV1 did not interact with CLV2, but can interact weakly with CRN. We also found that CLV1 can interact with CLV2-CRN heterodimers, implying that these three proteins may form a complex. Moreover, CRN, rather than CLV1 and CLV2, was able to form homodimers without CLV3 stimulation. Taken together, our results add direct evidence to the newly proposed two-parallel receptor pathways model and therefore provide new insights into the CLV3 signaling pathway.

Plant cell cultures provide an important method for production and supply of a variety of natural products, where conditions can be easily controlled, manipulated, and optimized. Development and optimization of plant cell culture processes require both bioprocess engineering and metabolic engineering approaches. Cultures are generally highly heterogeneous, with significant variability amongst cells in terms of growth, metabolism, and productivity of key metabolites. Taxus cultures produce the important anti-cancer agent Taxol((R)) (i.e., paclitaxel) and have demonstrated significant variability amongst cell populations in culture with regard to paclitaxel accumulation, cell cycle participation, and protein synthesis. To fully understand the link between cellular metabolism and culture behavior and to enable targeted metabolic engineering approaches, cultures need to be studied at a single cell level. This chapter describes the application of plant cell flow cytometric techniques to investigate culture heterogeneity at the single cell level, in order to optimize culture performance through targeted metabolic engineering. Flow cytometric analytical methods are described to study Taxus single cells, protoplasts, and nuclei suspensions with respect to secondary metabolite accumulation, DNA content, cell size, and complexity. Reproducible methods to isolate these single particle suspensions from aggregated Taxus cultures are discussed. Methods to stain both fixed and live cells for a variety of biological markers are provided to enable characterization of cell phenotypes. Fluorescence-activated cell sorting (FACS) methods are also presented to facilitate isolation of certain plant cell culture populations for both analysis and propagation of superior cell lines for use in bioprocesses.

Dedifferentiation signifies the capacity of somatic cells to acquire stem cell-like properties. This process can be induced during normal development and as a response to various stimuli, such as pathogen infection and wounding. Dedifferentiation also characterizes the transition of differentiated leaf cells into protoplasts (plant cells devoid of cell walls), a transition accompanied by widespread chromatin decondensation. Transcriptome profiling of dedifferentiating protoplast cells revealed striking similarities with senescing cells; both display a large increase in the expression of genes of specific transcription factor (TF) families, including ANAC, WRKY, bZIP, and C2H2. Further analysis showed that leaves induced to senesce by exposure to dark display characteristic features of dedifferentiating cells, including chromatin decondensation, disruption of the nucleolus, and condensation of rRNA genes. Considering that premature senescence can be induced by various stress conditions both in plant and animal cells, our results suggest that the response of plant and also animal cells to certain stresses converges on cellular dedifferentiation whereby cells first acquire stem cell-like state prior to acquisition of a new cell fate (e.g., reentry into the cell cycle or death).

The thylakoid membrane forms stacked thylakoids interconnected by 'stromal' lamellae. Little is known about the mobility of proteins within this system. We studied a stromal lamellae protein, Hcf106, by targeting an Hcf106-GFP fusion protein to the thylakoids and photobleaching. We find that even small regions fail to recover Hcf106-GFP fluorescence over periods of up to 3 min after photobleaching. The protein is thus either immobile within the thylakoid membrane, or its diffusion is tightly restricted within distinct regions. Autofluorescence from the photosystem II light-harvesting complex in the granal stacks likewise fails to recover. Integral membrane proteins within both the stromal and granal membranes are therefore highly constrained, possibly forming 'microdomains' that are sharply separated.

Although rational method and global technique have been successfully applied in strain improvement respectively, the demand for engineering complex phenotypes required combinatorial approach. The technology of genome shuffling has been presented as a novel whole genome engineering approach for the rapid improvement of cellular phenotypes. This approach using recursive protoplast fusion with multi-parental strains offers the advantage of recombination throughout the entire genome without the necessity for genome sequence data or network information. Genome shuffling has been demonstrated as an effective method, which is not only for producing improved strain but also for providing information on complex phenotype. In this review we attempt to present the advantage of genome shuffling, introduce the procedure of this technology, summarize the applications of this approach for phenotype improvement and then give perspective on the development of this method in the future.

Oxidative bursts from plants play significant roles in plant disease defense and signal transduction; however, it has not hitherto been investigated on individual living plant cells. In this article, we fabricated a novel sensitive electrochemical sensor based on electrochemical deposition of Pt nanoparticles on the surface of carbon fiber microdisk electrodes via a nanopores containing polymer matrix, Nafion. The numerous hydrophilic nanochannels in the Nafion clusters coated on the electrode surface served as the molecular template for the deposition and dispersion of Pt, which resulted in the uniform construction of small Pt nanoparticles. The novel sensor displayed a high sensitivity for detection of H(2)O(2) with a detection limit of 5.0 x 10(-9) M. With the use of this microelectrochemical sensor, the oxidative burst from individual living plant protoplasts have been real-time monitored for the first time. The results showed that oxidative burst from single protoplasts triggered by a pathogen analogue were characterized by quanta release with a large number of "transient oxidative microburst" events, and protoplasts from the transgenic plants biologically displayed better disease-resistance and showed a distinguished elevation and longer-lasting oxidative burst.

The last steps of the Leu biosynthetic pathway and the Met chain elongation cycle for glucosinolate formation share identical reaction types suggesting a close evolutionary relationship of these pathways. Both pathways involve the condensation of acetyl-CoA and a 2-oxo acid, isomerization of the resulting 2-malate derivative to form a 3-malate derivative, the oxidation-decarboxylation of the 3-malate derivative to give an elongated 2-oxo acid, and transamination to generate the corresponding amino acid. We have now analyzed the genes encoding the isomerization reaction, the second step of this sequence, in Arabidopsis thaliana. One gene encodes the large subunit and three encode small subunits of this enzyme, referred to as isopropylmalate isomerase (IPMI) with respect to the Leu pathway. Metabolic profiling of large subunit mutants revealed accumulation of intermediates of both Leu biosynthesis and Met chain elongation, and an altered composition of aliphatic glucosinolates demonstrating the function of this gene in both pathways. In contrast, the small subunits appear to be specialized to either Leu biosynthesis or Met chain elongation. Green fluorescent protein tagging experiments confirms the import of one of the IPMI small subunits into the chloroplast, the localization of the Met chain elongation pathway in these organelles. These results suggest the presence of different heterodimeric IPMIs in Arabidopsis chloroplasts with distinct substrate specificities for Leu or glucosinolate metabolism determined by the nature of the different small subunit.

The Anaphase Promoting Complex (APC) controls CDK activity by targeting the ubiquitin-dependent proteolysis of S-phase and mitosis-promoting cyclins. Here, we report that the ectopic expression of the Arabidopsis CDC27a, an APC subunit, accelerates plant growth and results in plants with increased biomass production. CDC27a overexpression was associated to apical meristem restructuration, protoplasts with higher (3)H-thimidine incorporation and altered cell-cycle marker expression. Total protein extracts immunoprecipitated with a CDC27a antibody showed ubiquitin ligase activity, indicating that the Arabidopsis CDC27a gets incorporated into APC complexes. These results indicate a role of AtCDC27a in regulation of plant growth and raise the possibility that the activity of the APC and the rates of plant cell division could be regulated by the concentration of the CDC27a subunit.

Nitrate, one of the major nitrogen sources for plants, is stored in the vacuole. Nitrate accumulation within the vacuole is primarily mediated by the NO(3)(-)/H(+) exchanger AtCLCa, which belongs to the chloride channel (CLC) family. Crystallography analysis of hCLC5 suggested that the C-terminal domain, composed by two cystathionine beta-synthetase motifs in all eukaryotic members of the CLC family is able to interact with ATP. However, interaction of nucleotides with a functional CLC protein has not been unambiguously demonstrated. Here we show that ATP reversibly inhibits AtCLCa by interacting with the C-terminal domain. Applying the patch clamp technique to isolated Arabidopsis thaliana vacuoles, we demonstrate that ATP reduces AtCLCa activity with a maximum inhibition of 60%. ATP inhibition of nitrate influx into the vacuole at cytosolic physiological nitrate concentrations suggests that ATP modulation is physiologically relevant. ADP and AMP do not decrease the AtCLCa transport activity; nonetheless, AMP (but not ADP) competes with ATP, preventing inhibition. A molecular model of the C terminus of AtCLCa was built by homology to hCLC5 C terminus. The model predicted the effects of mutations of the ATP binding site on the interaction energy between ATP and AtCLCa that were further confirmed by functional expression of site-directed mutated AtCLCa.

Protoplasts isolated from the mycobiont of a cultured lichen Usnea ghattensis were fused with protoplasts of the fungus Aspergillus nidulans in order to increase the growth rate of the cultured lichen mycobiont in vitro. The maximum protoplast yield (102 x 10(4)/g fresh cell mass) was reached in citrate buffer with 50 mmol/L 2-sulfanylethanol ('2-mercaptoethanol') containing 0.1 % Novozym after 1.5 h at pH 5 and

Caspase-like proteases have been demonstrated to be involved in plant programmed cell death (PCD). Here, the time scale of caspase-3-like protease activation was investigated in single living plant cells undergoing PCD induced by ultraviolet C (UV-C) overexposure. The real-time detection of caspase-3-like protease activation was achieved by measuring the degree of fluorescence resonance energy transfer (FRET) within a recombinant substrate containing enhanced cyan fluorescent protein (ECFP) linked by a peptide possessing the caspase-3 cleavage sequence, DEVD, to enhanced yellow fluorescent protein (EYFP; i.e. ECFP-DEVD-EYFP). Microscopic observations demonstrated that the ECFP-DEVD-EYFP fusion protein could be expressed correctly and the FRET from ECFP to EYFP could be found in transfected Arabidopsis (Arabidopsis thaliana) protoplasts. At 30 min after exposure to UV-C, caspase-3-like protease activation indicated by the decrease in FRET ratio occurred, taking about 1 h to reach completion in single living protoplasts. Mutation in the DEVD tag or a caspase-3 inhibitor could prevent the changes in FRET ratio induced by UV-C treatment, confirming that the decrease in FRET ratio was due to the cleavage of fusion protein as a result of caspase-3-like protease activation. This activation was further confirmed by in vitro caspase-3 substrate assay and western-blot analysis, showing the occurrence of cleavage in ECFP-DEVD-EYFP protein but not in the protein with a mutant DEVD tag. In summary, these results represent direct evidence for the activation of caspase-3-like protease in UV-C-induced PCD, and the FRET technique is a powerful tool for monitoring key events of PCD in living cells in real time.

This paper reports on the structural rearrangement of satellite DNA type I repeats and heterochromatin during the dedifferentiation and cell cycling of mesophyll protoplasts of cucumber (Cucumis sativus). These repeats were localized in the telomeric heterochromatin of cucumber chromosomes and in the chromocenters of interphase nuclei. The dramatic reduction of heterochromatin involves decondensation of subtelomeric repeats in freshly isolated protoplasts; however, there are not a great many remarkable changes in the expression profile. In spite of that, reformation of the chromocenters, occurring 48 h after protoplast isolation, is accompanied by recondensation of satellite DNA type I; however, only partial reassembly of these repeats was revealed. In this study, FISH and a flow cytometry assay show a correlation between the partial chromocenter and the repeats reassembly, and with the reentry of cultivated protoplasts into the cell cycle and first cell division. After that, divided cells displayed a higher variability in the expression profile than did leaves' mesophyll cells and protoplasts.

Guard cells are highly specialized cells that form tiny pores called stomata on the leaf surface. The opening and closing of stomata control leaf gas exchange and water transpiration as well as allow plants to quickly respond and adjust to new environmental conditions. Mesophyll cells are specialized for photosynthesis. Despite the phenotypic and obvious functional differences between the two types of cells, the full protein components and their functions have not been explored but are addressed here through a global comparative proteomics analysis of purified guard cells and mesophyll cells. With the use of isobaric tags for relative and absolute quantification (iTRAQ) tagging and two-dimensional liquid chromatography mass spectrometry, we identified 1458 non-redundant proteins in both guard cells and mesophyll cells of Brassica napus leaves. Based on stringent statistical criteria, a total of 427 proteins were quantified, and 74 proteins were found to be enriched in guard cells. Proteins involved in energy (respiration), transport, transcription (nucleosome), cell structure, and signaling are preferentially expressed in guard cells. We observed several well characterized guard cell proteins. By contrast, proteins involved in photosynthesis, starch synthesis, disease/defense/stress, and other metabolisms are preferentially represented in mesophyll cells. Of the identified proteins, 110 have corresponding microarray data obtained from Arabidopsis guard cells and mesophyll cells. About 72% of these proteins follow the same trend of expression at the transcript and protein levels. For the rest of proteins, the correlation between proteomics data and the microarray data is poor. This highlights the importance of quantitative profiling at the protein level. Collectively this work represents the most extensive proteomic description of B. napus guard cells and has improved our knowledge of the functional specification of guard cells and mesophyll cells.

In Arabidopsis, bric-a-brac, tramtrack and broad (BTB) domain scaffold proteins form a family of 80 proteins that have involvement in various signaling pathways. The five members of the subfamily of BTB AND TAZ DOMAIN proteins (BT1-BT5) have a typical domain structure that is only observed in land plants. Here, we present a functional analysis of the BT family, of which at least four members are encoded by auxin-responsive genes. BT1 is a short-lived protein that is characteristically targeted for degradation by the 26S proteasome. Expression pattern, gene structure and sequence analyses indicate that BT1 and BT2 are closely related. They both localize to the nucleus and the cytosol, whereas the remaining BT proteins were determined as cytosolic proteins. Detailed molecular and phenotypic analysis of plants segregating for null mutations in the BT family revealed substantial redundancy among the BT members, and highlighted that BT proteins perform crucial roles in both male and female gametophyte development. BT2 seems to be the predominant gene in this process, in which it is functionally replaced by BT3 and BT1 through reciprocal transcription regulation. Compensational expression alters the steady-state mRNA levels among the remaining BT family members when other BT members are lost, and this contributes towards functional redundancy. Our data provide a surprising example of functional redundancy among genes required during gametophyte development, something that could not be detected in the current screens for gametophyte mutants., (© 2009 The Authors. Journal compilation © 2009 Blackwell Publishing Ltd.)

To improve spinosyn-producing strain and enhance spinosyns yield, we studied the effects of glycin concentration and the operational time, temperature and lysozyme concentration on protoplast preparation of Saccharopolyspora spinosa SP06081. We also studied different regeneration media and osmotic stabilizing agents. In addition, we compared the change of morphology and spinosyns yield of the regenerated strains. The results showed that the Saccharopolyspora spinosa SP06081 protoplast yield was the highest under these conditions: the collected mycelium from SP06081 grown in Tryptic Soy Broth (TSB) medium with 0.2% glycin for 48 h was treated by 0.1 mg/mL lysozyme at 28 degrees C for 20 min, then plated on the R2YE medium with sucrose as osmotic stabilizer, the number of regeneration protoplast was up to 10(8)/mL. The protoplast-regenerated strains exhibited changes in morphology and antibiotic production, 29.3% protoplast-regenerated strains was characterized by loose mycelium and abundant broken branches as did their parent. Among them, 58.2% strains presented the trend to positive variation in spinosad yield, with the highest spinosad yield of up to 582.0 mg/L, 85.6% higher than that of their parent. There is significant correlation between the morphological differentiation and antibiotic yield of the protoplast-regenerated strains from spinosyn-producing strain.

Many of the structures involved in meiotic synapsis and recombination such as synaptonemal complexes (SCs) and recombination nodules (RNs) can be resolved only by electron microscopy. Therefore, electron microscopic (EM) immunolocalization using gold-conjugated antibodies is the best way to verify whether certain proteins are components of SCs or RNs. Here, we describe (1) preparing tomato primary microsporocyte protoplasts in leptotene, zygotene, and pachytene stages; (2) hypotonically bursting the protoplasts on glow-discharged glass and plastic-coated slides to make spreads of SCs; (3) immunolabeling proteins in SCs and RNs with colloidal gold; (4) staining SC spreads for EM; and (5) transferring SC spreads on plastic films to grids for EM.

L-forms are "cell wall-deficient" bacteria which are able to grow as spheroplasts or protoplasts. They can be differentiated into four types depending on their ability to revert to the parental, cell-walled form and to the extent of their cell-wall modification. L-forms are significant in modern science because of their contributions to an improved understanding of principal questions and their interactions with eukaryotes. This review particularly focuses on research using stable protoplast-type L-forms which have contributed to a better understanding of the structural and functional organisation of the cytoplasmic membrane and of cell division. These L-forms, which have only a single surrounding bilayer membrane, also represent a unique expression system for production of recombinant proteins. A large proportion of L-form publications concern their putative role in human disease and its therapy, a topic which is discussed briefly. L-forms have also been used to form intracellular associations with plant cells and have been shown to elicit induced disease resistance offering a novel method for plant protection. The recent decline in active research on L-forms is a concern as knowledge and experience, as well as unique L-form strains which have been maintained for decades, are being lost.

Polyphosphoinositides represent a minor group of phospholipids, accounting for less than 1% of the total. Despite their low abundance, these molecules have been implicated in various signalling and membrane trafficking events. Phosphatidylinositol 4-phosphate (PtdIns4P) is the most abundant polyphosphoinositide. (32)Pi-labelling studies have shown that the turnover of PtdIns4P is rapid, but little is known about where in the cell or plant this occurs. Here, we describe the use of a lipid biosensor that monitors PtdIns4P dynamics in living plant cells. The biosensor consists of a fusion between a fluorescent protein and a lipid-binding domain that specifically binds PtdIns4P, i.e. the pleckstrin homology domain of the human protein phosphatidylinositol-4-phosphate adaptor protein-1 (FAPP1). YFP-PH(FAPP1) was expressed in four plant systems: transiently in cowpea protoplasts, and stably in tobacco BY-2 cells, Medicago truncatula roots and Arabidopsis thaliana seedlings. All systems allowed YFP-PH(FAPP1) expression without detrimental effects. Two distinct fluorescence patterns were observed: labelling of motile punctate structures and the plasma membrane. Co-expression studies with organelle markers revealed strong co-labelling with the Golgi marker STtmd-CFP, but not with the endocytic/pre-vacuolar marker GFP-AtRABF2b. Co-expression with the Ptdins3P biosensor YFP-2 x FYVE revealed totally different localization patterns. During cell division, YFP-PH(FAPP1) showed strong labelling of the cell plate, but PtdIns3P was completely absent from the newly formed cell membrane. In root hairs of M. truncatula and A. thaliana, a clear PtdIns4P gradient was apparent in the plasma membrane, with the highest concentration in the tip. This only occurred in growing root hairs, indicating a role for PtdIns4P in tip growth.

Plant cells exhibit a lively activity of exocytosis and endocytosis at the plasma membrane. This is required for cell surface area regulation and also for membrane protein recycling. Although these processes are important for growth and development, they are understood purely with respect to their molecular mechanisms, regulation, spatial organization, and kinetics. According to the classical view, secretory vesicles release all their cargo in one step upon fusing and incorporating into the plasma membrane. Recent studies have shown that this model is oversimplifying the reality. Here we use real-time patch-clamp recording to monitor single-vesicle fusion and fission in order to resolve the kinetic properties of these elementary processes. The data show that single vesicles can, in a rhythmic fashion, make and break contact with the plasma membrane of plant protoplasts. Such oscillations are only possible if the two processes are linked by a distinct feedback system. The fact that similar rhythmic fusion/fission activity is also present in constitutive exocytosis in animal cells implies that the underlying mechanisms appear universal in eukaryotic cells.

Objective: To study the morphological changes of Cytophaga hutchinsonii cell during its life circle., Methods: Cytophaga hutchinsonii cell was observed under light microscope, fluorescence microscope and scanning electron microscope. The nucleoids in the cell were stained with fluorescent dye Hoechst33342 and examined by fluorescence microscopy., Results: We discovered that under starvation conditions, the long, flexible rod cell of Cytophaga hutchinsonii would bend and turn into circular cell. The circular cell failed to produce carboxymethyl cellulase. Some of the circular cells might further wind around and turn into tiny spherical cells. The tiny spherical cell similar to the microcyst of sporocytophaga could germ into long flexible rods again under certain circumstances. When growing cultures to logarithmic phase of cell growth, Cytophaga hutchinsonii cell with three nucleoids in it was occasionally observed, which indicated that the two strands of DNA might act differently in the initiation of DNA replication., Conclusion: This is the first detailed description of the formation process of circular cell and tiny spherical cell in the life circle of Cytophaga hutchinsonii. The result will help to further reveal the relation between morphologic change and cellulose degradation ability of the strain.

The B chain of ricin was expressed and delivered to the endoplasmic reticulum of tobacco protoplasts where it disappeared with time in a manner consistent with degradation. This turnover did not occur in the vacuoles or upon secretion. Indeed, several lines of evidence indicate that, in contrast to the turnover of endoplasmic reticulum-targeted ricin A chain in the cytosol, the bulk of expressed ricin B chain was degraded in the secretory pathway.