Proteome changes in Arabidopsis thaliana roots upon exposure to Cd2+

Cadmium is a major environmental pollutant that enters human food via accumulation in crop plants. Responses of plants to cadmium exposure--which directly influence accumulation rates--are not well understood. In general, little is known about stress-elicited changes in plants at the proteome level. Alterations in the root proteome of hydroponically grown Arabidopsis thaliana plants treated with 10 microM Cd(2+) for 24 h are reported here. These conditions trigger the synthesis of phytochelatins (PCs), glutathione-derived metal-binding peptides, shown here as PC2 accumulation. Two-dimensional gel electrophoresis using different pH gradients in the first dimension detected on average approximately 1100 spots per gel type. Forty-one spots indicated significant changes in protein abundance upon Cd(2+) treatment. Seventeen proteins found in 25 spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Selected results were independently confirmed by western analysis and selective enrichment of a protein family (glutathione S-transferases) through affinity chromatography. Most of the identified proteins belong to four different classes: metabolic enzymes such as ATP sulphurylase, glycine hydroxymethyltransferase, and trehalose-6-phosphate phosphatase; glutathione S-transferases; latex allergen-like proteins; and unknown proteins. These results represent a basis for reverse genetics studies to better understand plant responses to toxic metal exposure and to the generation of internal sinks for reduced sulphur.     

Arabidopsis group Ie formins localize to specific cell membrane domains, interact with actin-binding proteins and cause defects in cell expansion upon aberrant expression

The closely related proteins AtFH4 and AtFH8 represent the group Ie clade of Arabidopsis formin homologues. The subcellular localization of these proteins and their ability to affect the actin cytoskeleton were examined. AtFH4 protein activity was identified using fluorimetric techniques. Interactions between Arabidopsis profilin isoforms and AtFH4 were assayed in vitro and in vivo using pull-down assays and yeast-2-hybrid. The subcellular localization of group Ie formins was observed with indirect immunofluorescence (AtFH4) and an ethanol-inducible green fluorescent protein (GFP) fusion construct (AtFH8). AtFH4 protein affected actin dynamics in vitro, and yeast-2-hybrid assays suggested isoform-specific interactions with the actin-binding protein profilin in vivo. Indirect immunofluorescence showed that AtFH4 localized specifically to the cell membrane at borders between adjoining cells. Expression of an AtFH8 fusion protein resulted in GFP localization to cell membrane zones, similar to AtFH4. Furthermore, aberrant expression of AtFH8 resulted in the inhibition of root hair elongation. Taken together, these data suggest that the group Ie formins act with profilin to regulate actin polymerization at specific sites associated with the cell membrane.     

Effects of 2,4-D removal on the synthesis of specific proteins by Catharanthus roseus cell cultures

Total and neosynthesized proteins of periwinkle cell suspensions (Catharanthus roseus) were first investigated in cells grown in a 2,4-D-containing medium. Analysis of total (silver-stained) proteins by two-dimensional gel electrophoresis revealed that the levels of seventeen polypeptides were altered during the growth cycle of the cells. Analysis of in vivo [³⁵S]-methionine labeled polypeptides revealed differences in the synthesis of at least 35 polypeptides. Three polypeptides with molecular masses of 30, 35 and 39 kDa appeared to be specific markers of the early stationary phase. In a second sequence of experiments, cells were grown in a 2,4-D-free medium. Alterations in protein synthesis were observed: several polypeptides were expressed earlier in the 2,4-D-starved cells than in control cells; the synthesis of at least two specific polypeptides was increased in cells grown in 2,4-D-free medium, whereas the synthesis of three other polypeptides (molecular masses 33, 34 and 52.5 kDa) was switched on in these cells. As previous studies showed that 2,4-D depletion increased the alkaloid production in C. roseus cells, the present results may suggest that these polypeptides are implicated in the regulation of the alkaloid pathway.     

Characterization of photosystem I in rice (Oryza sativa L.) seedlings upon exposure to random positioning machine

To gain a better understanding of how photosynthesis is adapted under altered gravity forces, photosynthetic apparatus and its functioning were investigated in rice (Oryza sativa L.) seedlings grown in a random positioning machine (RPM). A decrease in fresh weight and dry weight was observed in rice seedlings grown under RPM condition. No significant changes were found in the chloroplast ultrastructure and total chlorophyll content between the RPM and control samples. Analyses of chlorophyll fluorescence and thermoluminescence demonstrate that PSII activity was unchanged under RPM condition. However, PSI activity decreased significantly under RPM condition. 77 K fluorescence emission spectra show a blue-shift and reduction of PSI fluorescence emission peak in the RPM seedlings. In addition, RPM caused a significant decrease in the amplitude of absorbance changes of P700 at 820 nm (A ₈₂₀) induced by saturated far-red light. Moreover, the PSI efficiency (Φ _I) decreased significantly under RPM condition. Immunoblot and blue native gel analyses further illustrate that accumulation of PSI proteins was greatly decreased in the RPM seedlings. Our results suggest that PSI, but not PSII, is down-regulated under RPM condition.     

Mutations in actin-related proteins 2 and 3 affect cell shape development in Arabidopsis

ACTIN-RELATED PROTEINS 2 and 3 form the major subunits of the ARP2/3 complex, which is known as an important regulator of actin organization in diverse organisms. Here, we report that two genes, WURM and DISTORTED1, which are important for cell shape control in Arabidopsis, encode the plant ARP2 and ARP3 orthologs, respectively. Mutations in these genes result in misdirected expansion of various cell types: trichome expansion is randomized, pavement cells fail to produce lobes, hypocotyl cells curl out of the normal epidermal plane, and root hairs are sinuous. At the subcellular level, cell shape changes are linked to severe filamentous actin aggregation and compromised vacuole fusion. Because all seven subunits of the ARP2/3 complex are present in plants, our data indicate that this complex may play a pivotal role during plant cell morphogenesis.     

Proteomic analysis of differentially expressed proteins in fungal elicitor-treated Arabidopsis cell cultures

Slow progress has been made in discovering plant genes governing the interaction of plant pathogens and their hosts using classical genetic approaches. Extensive studies employing DNA microarray techniques to identify global changes in gene expression during pathogen-host interaction have greatly enhanced discovery of genetic components regulating the plant defence response to pathogen attack. In this study, a complementary approach was used to identify changes in protein abundance during interaction of Arabidopsis cell cultures with a pathogen-derived elicitor. The soluble protein fractions were analysed by two-dimensional difference gel electrophoresis and proteins differentially expressed in response to treatment with fungal elicitor were identified via matrix-assisted laser desorption ionization-time of flight mass spectrometry. Elicitor responsive proteins included molecular chaperones, oxidative stress defence proteins, mitochondrial proteins, and enzymes of a diverse number of metabolic pathways. The findings, in combination with currently available microarray data, will form the basis of a filter to identify pivotal genes whose role in pathogen defence systems will require confirmation using gene knockout mutants.     

Methods for specific characterization of trace amounts of uridine nucleotides in animal cell cultures.

The use of specific indicator enzymes to characterize the nature of radioactive peaks, obtained from paper chromatograms of protein-free cell filtrates from cell cultures briefly incubated with [¹⁴C]galactose, has been described. The use of an internal standard ([³H]UDP-galactose) added to the filtrates before their exposure to the indicator enzymes illustrates the specificity of the method used, as well as its usefulness in quantitative analysis of the abundance of nucleotide sugars in cell cultures under different culture conditions.     

Time-course changes in fungal elicitor-induced lignan synthesis and expression of the relevant genes in cell cultures of Linum album

Linum album has been shown to accumulate anti-tumor podophyllotoxin (PTOX) and its related lignans. In the present study, we examined the effects of five fungal extracts on the production of lignans in L. album cell cultures. Fusarium graminearum extract induced the highest increase of PTOX [140μgg(-1) dry weight (DW) of the L. album cell culture] which is seven-fold greater than the untreated control, while Rhizopus stolonifer extract enhanced the accumulation of lariciresinol, instead of PTOX, up to 365μgg(-1) DW, which was 8.8-fold greater than the control. Quantitative PCR analyses showed that expression of the enzyme genes responsible for the PTOX biosynthesis cascade, such as pinoresinol-lariciresinol reductase (PLR), phenylalanine ammonia-lyase (PAL), cinnamoyl-CoA reductase (CCR) and cinnamyl-alcohol dehydrogenase (CAD) genes, were also up-regulated in a fungal extract-selective fashion. These results provide evidence that the fungal extracts used in this study differentially increase the production of PTOX or larisiresinol via the up-regulation of the genes in lignan biosynthesis in L. album cell cultures, and suggest that such selective actions of fungal elicitors on the lignan synthesis will lead to more efficient metabolic engineering-based production of PTOX and other beneficial lignans using L. album cell cultures.     

Identification by 2-D DIGE of apoplastic proteins regulated by oligogalacturonides in Arabidopsis thaliana

Oligogalacturonides (OGs) are elicitors of plant defence responses released from the homogalacturonan of the plant cell wall during the attack by pathogenic micro-organisms. The signalling pathway mediated by OGs remains poorly understood, and no proteins involved in their signal perception and transduction have yet been identified. In order to shed light into the molecular pathways regulated by OGs, a differential proteomic analysis has been carried out in Arabidopsis. Proteins from the apoplastic compartment were isolated and their expression compared between control and OG-treated seedlings. 2-D gels and difference in gel electrophoresis (DIGE) techniques were used to compare control and treated proteomes in the same gel. The analysis of subcellular proteomes from seedlings allowed the identification of novel and low abundance proteins that otherwise remain masked when total cellular extracts are investigated. The DIGE technique showed to be a powerful tool to overcome the high interexperiment variation of 2-D gels. Differentially expressed apoplastic proteins were identified by MS and included proteins putatively involved in recognition as well as proteins whose PTMs are regulated by OGs. Our findings underscore the importance of cell wall as a source of molecules playing a role in the perception of pathogens and provide candidate proteins involved in the response to OGs.     

A new type of compartment, defined by plant-specific Atg8-interacting proteins, is induced upon exposure of Arabidopsis plants to carbon starvation

Atg8 is a central protein in bulk starvation-induced autophagy, but it is also specifically associated with multiple protein targets under various physiological conditions to regulate their selective turnover by the autophagy machinery. Here, we describe two new closely related Arabidopsis thaliana Atg8-interacting proteins (ATI1 and ATI2) that are unique to plants. We show that under favorable growth conditions, ATI1 and ATI2 are partially associated with the endoplasmic reticulum (ER) membrane network, whereas upon exposure to carbon starvation, they become mainly associated with newly identified spherical compartments that dynamically move along the ER network. These compartments are morphologically distinct from previously reported spindle-shaped ER bodies and, in contrast to them, do not contain ER-lumenal markers possessing a C-terminal HDEL sequence. Organelle and autophagosome-specific markers show that the bodies containing ATI1 are distinct from Golgi, mitochondria, peroxisomes, and classical autophagosomes. The final destination of the ATI1 bodies is the central vacuole, indicating that they may operate in selective turnover of specific proteins. ATI1 and ATI2 gene expression is elevated during late seed maturation and desiccation. We further demonstrate that ATI1 overexpression or suppression of both ATI1 and ATI2, respectively, stimulate or inhibit seed germination in the presence of the germination-inhibiting hormone abscisic acid.     

Role of 2,4-dichlorophenoxyacetic acid (2,4-D) in somatic embryogenesis on cultured zygotic embryos of Arabidopsis: cell expansion, cell cycling, and morphogenesis during continuous exposure of embryos to 2,4-D

The relationship between cell expansion and cell cycling during somatic embryogenesis was studied in cultured bent-cotyledon-stage zygotic embryos of a transgenic stock of Arabidopsis thaliana harboring a cyclin 1 At:β-glucuronidase (GUS) reporter gene construct. In embryos cultured in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), following a brief period of growth by cell expansion, divisions were initiated in the procambial cells facing the adaxial side at the base of the cotyledons. Cell division activity later spread to almost the entire length of the cotyledons to form a callus on which globular and heart-shaped embryos appeared in about 10 d after culture. Anatomical and morphogenetic changes observed in cultured embryos were correlated with patterns of cell cycling by histochemical detection of GUS-expressing cells. Although early-stage somatic embryos did not develop further during their continued growth in the auxin-containing medium, maturation of embryos ensued upon their transfer to an auxin-free medium. In a small number of cultured zygotic embryos the shoot apical meristem was found to differentiate a leaf, a green tubular structure, or a somatic embryo. Contrary to the results from previous investigations, which have assigned a major role for the shoot apical meristem and cells in the axils of cotyledons in the development of somatic embryos on cultured zygotic embryos of A. thaliana, the present work shows that somatic embryos originate almost exclusively on the callus formed on the cotyledons. Other observations such as the induction of somatic embryos on cultured cotyledons and the inability of the embryo axis (consisting of the root, hypocotyl, and shoot apical meristem without the cotyledons) to form somatic embryos, reaffirm the important role of the cotyledons in somatic embryogenesis in this plant.     

Glutathione,glutathione disulfide,and S-glutathionylated proteins in cell cultures

The analysis of the global thiol–disulfide redox status in tissues and cells is a challenging task since thiols and disulfides can undergo artificial oxido-reductions during sample manipulation. Because of this, the measured values, in particular for disulfides, can have a significant bias. Whereas this methodological problem has already been addressed in samples of red blood cells and solid tissues, a reliable method to measure thiols and disulfides in cell cultures has not been previously reported.Here, we demonstrate that the major artifact occurring during thiol and disulfide analysis in cultured cells is represented by glutathione disulfide (GSSG) and S-glutathionylated proteins (PSSG) overestimation, due to artificial oxidation of glutathione (GSH) during sample manipulation, and that this methodological problem can be solved by the addition of N-ethylmaleimide (NEM) immediately after culture medium removal. Basal levels of GSSG and PSSG in different lines of cultured cells were 3–5 and 10–20 folds higher, respectively, when the cells were processed without NEM. NEM pre-treatment also prevented the artificial reduction of disulfides that occurs during the pre-analytical phase when cells are exposed to an oxidant stimulus. In fact, in the absence of NEM, after medium removal, GSH, GSSG and PSSG levels restored their initial values within 15–30 min, due to the activity of reductases and the lack of the oxidant. The newly developed protocol was used to measure the thiol–disulfide redox status in 16 different line cells routinely used for biomedical research both under basal conditions and after treatment with disulfiram, a thiol-specific oxidant (0–200 μM concentration range).Our data indicate that, in most cell lines, treatment with disulfiram affected the levels of GSH and GSSG only at the highest concentration. On the other hand, PSSG levels increased significantly also at the lower concentrations of the drug, and the rise was remarkable (from 100 to 1000 folds at 200 μM concentration) and dose-dependent for almost all the cell lines. These data support the suitability of the analysis of PSSG in cultured cells as a biomarker of oxidative stress.     

Planar arrangement of eukaryotic cells in merged hydrogels combines the advantages of 3-D and 2-D cultures

We report an unordered 2-D array of eukaryotic cells completely embedded in a 3-D matrix. Every cell is located at the same distance from the gel surface, which ensures uniformity of growth conditions and ease of observation characteristic of a 2-D culture. Each cell is firmly immobilized, and each has a unique address in the array. The cells can be rapidly screened, individually monitored during extended time periods, and cultured with the formation of spheroid microcolonies characteristic of a 3-D culture. Individual microcolonies can be extracted from the gel and further propagated, thus enabling isolation of pure cell clones from rather dense cell populations and rapid drug-free generation of stable cell lines.     

Effects of cell cycle specific exposure to 3H-thymidine or 3H-arginine on development and cell proliferation of mouse embryos

One-cell mouse embryos were exposed to either ³H-thymidine (100 or 200 kBq/ml) or ³H-arginine (2.5 to 50 kBq/ml) for 2 h either in G₁, S or G₂ phase. ³H-Arginine affected embryonic development and cell proliferation in an activity-dependent way irrespective of the cell cycle stage exposed, whereas ³H-thymidine was effective only at higher activities and only after exposure during S phase. Received: 12 July 1996 / Accepted in revised form: 30 September 1996     

Characterization of cytokinin and adenine transport in Arabidopsis cell cultures

Cytokinins are distributed through the vascular system and trigger responses of target cells via receptor-mediated signal transduction. Perception and transduction of the signal can occur at the plasma membrane or in the cytosol. The signal is terminated by the action of extra- or intracellular cytokinin oxidases. While radiotracer studies have been used to study transport and metabolism of cytokinins in plants, little is known about the kinetic properties of cytokinin transport. To provide a reference dataset, radiolabeled trans-zeatin (tZ) was used for uptake studies in Arabidopsis (Arabidopsis thaliana) cell culture. Uptake kinetics of tZ are multiphasic, indicating the presence of both low- and high-affinity transport systems. The protonophore carbonyl cyanide m-chlorophenylhydrazone is an effective inhibitor of cytokinin uptake, consistent with H(+)-mediated uptake. Other physiological cytokinins, such as isopentenyl adenine and benzylaminopurine, are effective competitors of tZ uptake, whereas allantoin has no inhibitory effect. Adenine competes for zeatin uptake, indicating that the degradation product of cytokinin oxidases is transported by the same systems. Comparison of adenine and tZ uptake in Arabidopsis seedlings reveals similar uptake kinetics. Kinetic properties, as well as substrate specificity determined in cell cultures, are compatible with the hypothesis that members of the plant-specific purine permease family play a role in adenine transport for scavenging extracellular adenine and may, in addition, be involved in low-affinity cytokinin uptake.