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.     

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.     

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.     

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.     

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.     

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     

CDK2 differentially controls normal cell senescence and cancer cell proliferation upon exposure to reactive oxygen species

The linker of nucleus and cytoskeleton (LINC) complex, including nesprin-1, has been suggested to be crucial for many biological processes. Previous studies have shown that mutations in nesprin-1 cause abnormal cellular functions and diseases, possibly because of insufficient force transmission to the nucleus through actin filaments (F-actin) bound to nesprin-1. However, little is known regarding the mechanical interaction between the nucleus and F-actin through nesprin-1. In this study, we examined nuclear deformation behavior in nesprin-1 knocked-down endothelial cells (ECs) subjected to uniaxial stretching by evaluating nuclear strain from lateral cross-sectional images. The widths of nuclei in nesprin-1 knocked-down ECs were smaller than those in wild-type cells. In addition, nuclear strain in nesprin-1 knocked-down cells, which is considered to be compressed by the actin cortical layer, increased compared with that in wild-type cells under stretching condition. These results indicate that nesprin-1 knockdown releases the nucleus from the tension of F-actin bound to the nucleus, thereby increasing allowance for deformation before stretching, and that F-actin bound to the nucleus through nesprin-1 causes sustainable force transmission to the nucleus.     

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.     

Autophagy deficiency leads to accumulation of ubiquitinated proteins,ER stress,and cell death in Arabidopsis

Autophagy is a homeostatic degradation and recycling process that is also involved in defense against microbial pathogens and in certain forms of cellular suicide. Autophagy has been proposed to negatively regulate plant immunity-associated cell death related to the hypersensitive response (HR), as older autophagy-deficient mutants are unable to contain this type of cell death 5 to 10 d after infection. Such spreading cell death was found to require NPR1 (nonexpressor of PR genes 1), but surprisingly did not occur in younger atg mutants. In contrast, we find that npr1 mutants are not impaired in rapid programmed cell death activation upon pathogen recognition. Furthermore, our molecular evidence suggests that the NPR1-dependent spreading cell death in older atg mutants may originate from an inability to cope with excessive accumulation of ubiquitinated proteins and ER stress which derive from salicylic acid (SA)-dependent signaling (e.g., systemic acquired resistance). We also demonstrate that both senescence and immunity-related cell death seen in older atg mutants can be recapitulated in younger atg mutants primed with ER stress. We therefore propose that the reduction in SA signaling caused by npr1 loss-of-function is sufficient to alleviate the stress levels accumulated during aging in autophagy deficient cells which would otherwise become insurmountable and lead to uncontrolled cell death.     

Proteomic analysis of changes in the extracellular matrix of Arabidopsis cell suspension cultures induced by fungal elicitors

A proteomic approach has been applied to investigate changes in the extracellular matrix of Arabidopsis thaliana cell suspension cultures following treatments with two fungal pathogen elicitors, chitosan and extracts of Fusarium moniliforme. The oxidative burst and induction of glutathione S-transferase were used as markers for induction of the pathogen defence response. Changes in the cell wall and culture filtrate proteome were profiled. Proteins whose abundance changed reproducibly were analysed via matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry (MS). An increase in the level of two classical cell wall proteins (a putative endochitinase and a polygalacturonase inhibiting protein) and two novel proteins (a putative receptor-like protein kinase and a probable apospory-associated protein) were seen at 24 hours following elicitation. The level of an unknown protein and a hypothetical protein, which has some homology to serine carboxypeptidases, were decreased at 24 hours post-elicitation. In the culture filtrate extracts, we identified two pathogen elicitor responsive proteins, a xyloglucan endo-1,4-beta-D glucanases (XEG) and a peroxidase. Using a combination of two-dimensional polyacrylamide gel electrophoresis, immunoblotting with a phosphotyrosine-specific antibody, and MALDI-TOF MS we discovered that spots that represent putative lectin receptor-like kinase, a putative endochitinase and a XEG possess phosphorylated tyrosine residues. The identification of phosphorylated bona fide cell wall proteins and a putative extracellular receptor-like kinase with no transmembrane domain implicate the existence of an extracellular phosphorylation network which could be involved in intercellular communication.