Arabidopsis Thaliana Suspension Cultures Research Articles

Spermine and spermidine inhibit or induce programmed cell death in Arabidopsis thaliana in vitro and in vivo in a dose-dependent manner.

Polyamines are ubiquitous biomolecules with a number of established functions in eukaryotic cells. In plant cells, polyamines have previously been linked to abiotic and biotic stress tolerance, as well as to the modulation of programmed cell death (PCD), with contrasting reports on their pro-PCD and pro-survival effects. Here, we used two well-established platforms for the study of plant PCD, Arabidopsis thaliana suspension cultures cells and the root hair assay, to examine the roles of the polyamines spermine and spermidine in the regulation of PCD. Using these systems for precise quantification of cell death rates, we demonstrate that both polyamines can trigger PCD when applied exogenously at higher doses, whereas at lower concentrations they inhibit PCD induced by both biotic and abiotic stimuli. Furthermore, we show that concentrations of polyamines resulting in inhibition of PCD generated a transient ROS burst in our experimental system, and activated the expression of oxidative stress- and pathogen response-associated genes. Finally, we examined PCD responses in existing Arabidopsis polyamine synthesis mutants, and identified a subtle PCD phenotype in Arabidopsis seedlings deficient in thermo-spermine. The presented data show that polyamines can have a role in PCD regulation; however, that role is dose-dependent and consequently they may act as either inhibitors, or inducers, of PCD in Arabidopsis.

Cooperative recruitment of RDR6 by SGS3 and SDE5 during small interfering RNA amplification in Arabidopsis

Small interfering RNAs (siRNAs) are often amplified from transcripts cleaved by RNA-induced silencing complexes (RISCs) containing a small RNA (sRNA) and an Argonaute protein. Amplified siRNAs, termed secondary siRNAs, are important for reinforcement of target repression. In plants, target cleavage by RISCs containing 22-nucleotide (nt) sRNA and Argonaute 1 (AGO1) triggers siRNA amplification. In this pathway, the cleavage fragment is converted into double-stranded RNA (dsRNA) by RNA-dependent RNA polymerase 6 (RDR6), and the dsRNA is processed into siRNAs by Dicer-like proteins. Because nonspecific RDR6 recruitment causes nontarget siRNA production, it is critical that RDR6 is specifically recruited to the target RNA that serves as a template for dsRNA formation. Previous studies showed that Suppressor of Gene Silencing 3 (SGS3) binds and stabilizes 22-nt sRNA-containing AGO1 RISCs associated with cleaved target, but how RDR6 is recruited to targets cleaved by 22-nt sRNA-containing AGO1 RISCs remains unknown. Here, using cell-free extracts prepared from suspension-cultured Arabidopsis thaliana cells, we established an in vitro system for secondary siRNA production in which 22-nt siRNA-containing AGO1-RISCs but not 21-nt siRNA-containing AGO1-RISCs induce secondary siRNA production. In this system, addition of recombinant Silencing Defective 5 (SDE5) protein remarkably enhances secondary siRNA production. We show that RDR6 is recruited to a cleavage fragment by 22-nt siRNA-containing AGO1-RISCs in coordination with SGS3 and SDE5. The SGS3-SDE5-RDR6 multicomponent recognition system and the poly(A) tail inhibition may contribute to securing specificity of siRNA amplification.

Sodium transport by endocytic vesicles in cultured Arabidopsis thaliana (L.) Heynh. cells

The involvement of endocytosis in Na+ internalization by suspension-cultured Arabidopsis thaliana (L.) Heynh. cells under salt stress was investigated. Using epifluorescence and confocal laser-scanning microscopy in co-localization experiments with a marker of the endocytic structures, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl) hexatrienyl) pyridinium dibromide (FM 4-64), and a membrane-impermeable Na+ indicator, Asante Natrium Green-2 tetramethylammonium salt (ANG-2 TMA), intracellular Na+ ion uptake via endocytosis, but not through the plasma-membrane ion channels or transporters, was detected. The punctate fluorescence of the two markers was co-localized in the microvacuoles (MVs) with sizes up to 5 μm. The study of cell ultrastructure by transmission electron microscopy revealed the fusion of MVs into larger ones during an incubation of the cells in medium containing 100 mM NaCl. Wortmannin, an inhibitor of multivesicular body/late endosome/pre-vacuolar compartment fusion with vacuoles, substantially lowered intracellular Na+ content. Apparently, endocytic Na+ uptake and subsequent fusion of Na+-containing membrane structures provided direct transport of external Na+ ions into the vacuoles, bypassing the cytosol.

Tandem affinity purification of AtTERT reveals putative interaction partners of plant telomerase in vivo.

The life cycle of telomerase involves dynamic and complex interactions between proteins within multiple macromolecular networks. Elucidation of these associations is a key to understanding the regulation of telomerase under diverse physiological and pathological conditions from telomerase biogenesis, through telomere recruitment and elongation, to its non-canonical activities outside of telomeres. We used tandem affinity purification coupled to mass spectrometry to build an interactome of the telomerase catalytic subunit AtTERT, using Arabidopsis thaliana suspension cultures. We then examined interactions occurring at the AtTERT N-terminus, which is thought to fold into a discrete domain connected to the rest of the molecule via a flexible linker. Bioinformatic analyses revealed that interaction partners of AtTERT have a range of molecular functions, a subset of which is specific to the network around its N-terminus. A significant number of proteins co-purifying with the N-terminal constructs have been implicated in cell cycle and developmental processes, as would be expected of bona fide regulatory interactions and we have confirmed experimentally the direct nature of selected interactions. To examine AtTERT protein-protein interactions from another perspective, we also analysed AtTERT interdomain contacts to test potential dimerization of AtTERT. In total, our results provide an insight into the composition and architecture of the plant telomerase complex and this will aid in delineating molecular mechanisms of telomerase functions.

Proteomic Analysis of Microtubule Interacting Proteins over the Course of Xylem Tracheary Element Formation in Arabidopsis

Plant vascular cells, or tracheary elements (TEs), rely on circumferential secondary cell wall thickenings to maintain sap flow. The patterns in which TE thickenings are organized vary according to the underlying microtubule bundles that guide wall deposition. To identify microtubule interacting proteins present at defined stages of TE differentiation, we exploited the synchronous differentiation of TEs in Arabidopsis thaliana suspension cultures. Quantitative proteomic analysis of microtubule pull-downs, using ratiometric (14)N/(15)N labeling, revealed 605 proteins exhibiting differential accumulation during TE differentiation. Microtubule interacting proteins associated with membrane trafficking, protein synthesis, DNA/RNA binding, and signal transduction peaked during secondary cell wall formation, while proteins associated with stress peaked when approaching TE cell death. In particular, CELLULOSE SYNTHASE-INTERACTING PROTEIN1, already associated with primary wall synthesis, was enriched during secondary cell wall formation. RNAi knockdown of genes encoding several of the identified proteins showed that secondary wall formation depends on the coordinated presence of microtubule interacting proteins with nonoverlapping functions: cell wall thickness, cell wall homogeneity, and the pattern and cortical location of the wall are dependent on different proteins. Altogether, proteins linking microtubules to a range of metabolic compartments vary specifically during TE differentiation and regulate different aspects of wall patterning.

The plant cell inhibitor KRP6 is involved in multinucleation and cytokinesis disruption in giant-feeding cells induced by root-knot nematodes

The plant cell cycle inhibitor gene KRP6 has been investigated in roots infected by plant-parasitic root-knot nematodes (Meloidogyne spp.). Unexpectedly, KRP6 overexpressing lines revealed a distinct role for this specific KRP as an activator of the mitotic cell cycle. This function was confirmed in Arabidopsis thaliana suspension cultures ectopically expressing KRP6. A blockage in the mitotic exit was observed in cell suspensions and in giant cells resulted in the appearance of multi-nucleated cells. KRP6 expression during nematode infection and the similarity in phenotypes among KRP6 overexpressing cell cultures and giant-cell morphology strongly suggest that KRP6 is involved in multinucleation and acytokinesis occurring in giant-cells. Once again nematodes have been shown to manipulate the plant cell cycle machinery in order to promote gall establishment.

Atomic Force Microscopy Stiffness Tomography on Living Arabidopsis thaliana Cells Reveals the Mechanical Properties of Surface and Deep Cell-Wall Layers during Growth

Cell-wall mechanical properties play a key role in the growth and the protection of plants. However, little is known about genuine wall mechanical properties and their growth-related dynamics at subcellular resolution and in living cells. Here, we used atomic force microscopy (AFM) stiffness tomography to explore stiffness distribution in the cell wall of suspension-cultured Arabidopsis thaliana as a model of primary, growing cell wall. For the first time that we know of, this new imaging technique was performed on living single cells of a higher plant, permitting monitoring of the stiffness distribution in cell-wall layers as a function of the depth and its evolution during the different growth phases. The mechanical measurements were correlated with changes in the composition of the cell wall, which were revealed by Fourier-transform infrared (FTIR) spectroscopy. In the beginning and end of cell growth, the average stiffness of the cell wall was low and the wall was mechanically homogenous, whereas in the exponential growth phase, the average wall stiffness increased, with increasing heterogeneity. In this phase, the difference between the superficial and deep wall stiffness was highest. FTIR spectra revealed a relative increase in the polysaccharide/lignin content.

<i>N</i>-Glycan structures of glycoproteins in suspension-cultured <i>Arabidopsis thaliana</i> T87 cells

Suspension-cultured plant cells are feasible bioreactors for the production of pharmaceutical proteins. Post-translational modification, such as glycosylation, is important for in vivo biological and physiological roles of proteins. However, little is known about the effect of suspension-culture conditions on protein N-glycosylation. In this study, we studied the N-glycan structures of soluble and endogenous glycoproteins at different growth stages of suspension-cultured Arabidopsis thaliana T87 cells. At the late growth stage, the amount of N-glycans with terminal N-acetylglucosamine (GlcNAc) decreased, whereas that of N-glycans with fucose and xylose increased. This indicates that N-glycans have more plant-specific sugar residues as the cultivation reaches the late or end stage of plant growth.

Isolation of Daucus carota ethylene insensitive3-like (DcEIL) involved in stress-inducible DcMYB1 expression in suspension-cultured carrot cells

In carrot suspension-cultured cells, the expression of phenylalanine ammonia-lyase 1 (DcPAL1) gene was crucially regulated by the transcription regulatory factor Daucus carota MYB1 (DcMYB1). To elucidate the regulatory mechanism of DcMYB1 expression, we isolated and identified the transcription regulatory factor D. carota ethylene-insensitive3 (EIN3)-like protein (DcEIL) from a cDNA library prepared from suspension-cultured carrot cells treated with the “dilution effect” using a yeast one-hybrid system. DcEIL bound to a region of the DcMYB1 promoter containing a putative cis-element, which suppressed DcMYB1 promoter expression. The amino acid sequence of DcEIL contained the predicted signal sequence for nuclear localization, and transportation to the nucleus was confirmed using a green fluorescent protein-DcEIL fusion protein expressed in suspension-cultured Arabidopsis thaliana cells. The DcEIL gene was constitutively expressed irrespective of the elicitor treatment or the dilution effect. These results suggest that the binding of DcEIL to a cis-element of the DcMYB1 promoter might be regulated at the posttranslational level as a consequence of the regulation of DcPAL1 gene expression via DcMYB1 expression.

Phosphate levels and expression of phosphoribosylpyrophosphate synthetase isozymes in suspension-cultured Arabidopsis thaliana cells

In plants, as well as the generally distributed phosphate-dependent phosphoribosylpyrophosphate (PRPP) synthetase (class I), phosphate-independent PRPP synthetase (class II) is also present. To investigate the effect of the inorganic phosphate (Pi) level on the two classes of PRPP synthetase, we first estimated the cellular phosphate level using 33Pi in suspension-cultured Arabidopsis thaliana cells. 33Pi in the culture medium was taken up by the cells, and the concentration of Pi in cells increased up to 5.5 μmol/g fresh weight within 24 h once the cells were transferred to the fresh medium; its concentration then fell because of the conversion of Pi to organic compounds. In vitro activity of PRPP synthetase increased after inoculation and maintained a high activity until the early exponential growth stage. The transcript levels of PRS1 and PRS2 encoding class I and PRS3 encoding class II enzymes increased rapidly after the cells were transferred to the fresh medium, then remained almost constant during the early exponential growth phase. In contrast, constitutive expression of PRS4 encoding cytosolic class II enzyme was observed during culture. During long-term Pi-starvation the transcript levels of PRS1 and PRS2 were reduced, but PRS3 and PRS4 were expressed continually during the Pi-starvation. Pi-dependent PRPP synthetase activity was simultaneously reduced, but Pi-independent activity did not change. Expression of PRS1 and PRS2 and the activity of Pi-dependent enzyme grew to normal rates by 24 h after supply of Pi.

Expression of Glucose-6-phosphate Dehydrogenase and 6-Phosphogluconate Dehydrogenase Isoform Genes in Suspension-Cultured Arabidopsis thaliana Cells

The activities of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH, EC 1.1.1.44) were found to increase in suspension-cultured Arabidopsis thaliana cells after 10-day-old stationary phase cells were transferred to fresh Murashige-Skoog medium. The activities of these enzymes peaked early in the exponential growth stage of the culture (day 4) and then decreased gradually. The transcript levels of six isoform genes of G6PDH (AtG6PDI to AtG6PD6) and three of 6PGDH (At6PGD1 to At6PGD3) were monitored during the culture. Two distinct transcript accumulation patterns were observed. In type A, the level of transcripts increased rapidly one day after the cells were inoculated into the fresh medium, and then remained almost constant until the culture reached its stationary phase (day 7). In type B, the transcripts were accumulated transiently at the first day after cell inoculation, then promptly decreased. We also investigated the effect of phosphate (Pi)-starvation and recovery on the expression of these genes. For this, the early stationary phase cultures (day 7) were transferred to fresh Pi-free culture medium. During 7 days of phosphate starvation, no growth of cultures was observed, and the transcript levels of all G6PDH and 6PGDH isoform genes were reduced, apart from one G6PDH isoform gene, AtG6PD5, which was continuously expressed throughout Pi-starvation. Compared to the reduction of almost all isoform genes of G6PDH in Pi-starved cultures, the reduction of 6PGDH genes was less severe. We discuss the localization and possible role of individual isoform genes of G6PDH and 6PGDH in connection with published databases.

Inhibitors of protein glycosylation or secretion change the pattern of extracellular proteins in suspension-cultured cells of Arabidopsis thaliana

Cell walls are essential for plant development and morphogenesis. The majority of wall proteins are glycosylated, either as N- or O-glycans. Various inhibitors of glycosylation and secretion are used to determine the importance of wall proteins for the functioning of the walls. Tunicamycin is an inhibitor of the first enzyme in the N-glycosylation pathway, 3,4-dehydroproline inhibits peptidyl proline hydroxylation, and Brefeldin A is an inhibitor of vesicle trafficking, disrupting the delivery of wall polymers to the apoplast. In inhibitor-treated suspension-cultured Arabidopsis thaliana cells, qualitative and quantitative differences in the extracellular proteome were observed for both proteins secreted into medium or ionically-bound in the walls. Lack of O-glycosylation resulted in the selective protein loss from the extracellular compartments. Following tunicamycin treatment the secretion of additional proteins as well as ER-resident chaperones from the Hsp70 and Hsp90 families outside the protoplasts was noted. Moreover, changes in the proteolytic degradation pattern of culture filtrate proteins were also observed. Application of Brefeldin A resulted in transient and selective loss of individual proteins from the extracellular compartments of A. thaliana cell suspension. We conclude that post-translational modifications are vital for the proper functioning of wall proteins. N-glycosylation is crucial for their proper folding and stability. Extracellular compartments could also serve as a sink for improperly folded proteins during the unfolded protein response.

A diffusible signal from germinating Orobanche ramosa elicits early defense responses in suspension-cultured Arabidopsis thaliana

In plant/parasitic plant interaction, little is known about the host plant response before the establishment of the parasite within the host. In the present work, we focused on host responses to parasitic plant, O. ramosa in the early stage of infection. We used a co-culture system of A. thaliana suspension cells and O. ramosa germinated-seeds to avoid parasite attachment. We showed that O. ramosa induced H2O2 generation and camalexin synthesis by A. thaliana followed by a drastic increase in cell death. We further demonstrated that a heat sensitive diffusible signal is responsible for this cell death. These data indicate that recognition of O. ramosa occurs before the attachment of the parasite and initiates plant defence responses.

N-linked glycan structures of glycoproteins in suspension-cultured Arabidopsis thaliana MM2d cells

The oligosaccharide structures of total glycoproteins in suspension-cultured Arabidopsis thaliana MM2d cells are determined. The N-linked sugar chains released by hydrazinolysis were labeled with 2-aminopyridine, and analyzed by a combination of HPLC, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and exoglucosidase digestion. The glycan structures determined were GlcNAcMan3FucXylGlcNAc2 (8.5%), Man3FucXylGlcNAc2 (70.0%), Man3XylGlcNAc2 (13.7%), and GlcNAcMan3XylGlcNAc2 (7.8%). All the glycan structures of glycoproteins from Arabidopsis MM2d cells used in this study contain typical plant bisecting either b1,2-xylose or a1,3-fucose residues, showing that suspension-cultured MM2d cells have a glycosylation potential similar to that of tobacco BY2 suspension- cultured cells.

Transcriptional analysis of calcium-dependent and calcium-independent signalling pathways induced by oligogalacturonides

alpha-1,4-linked oligogalacturonides (OGs) are pectic fragments of plant cell walls that are able to induce defence and developmental responses. To understand plant responses to OGs at the transcriptional level, changes in gene expression were examined using oligonucleotide-based microarrays that cover almost the entire Arabidopsis transcriptome. In suspension-cultured Arabidopsis thaliana (L.) Columbia hypocotyl cells, approximately 4% of the total transcriptome exhibited significant change in abundance in response to treatment with OGs for 2 h. Steady-state changes in the abundance of transcripts encoding stress- and disease-related proteins, signalling components, and transcription factors were particularly noteworthy. As in other plant cell types, OGs elicit a rapid, but transient, elevation in cytosolic free Ca(2+). The Ca(2+) transient can be abolished by the protein kinase inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) and by the Ca(2+) channel inhibitor La(3+), thereby facilitating a distinction between Ca(2+)-dependent and -independent transcriptional responses. Among the 244 transcripts that were up-regulated by OGs, the response of 93 (38%) was selectively sensitive to abolition of the Ca(2+) transient. These OG-up-regulated, Ca(2+)-dependent transcripts included two noteworthy classes, the first comprising genes involved in cell wall modification following pathogen attack, and the second consisting of genes involved in the biosynthesis of jasmonate and C6 volatile compounds. These results support the notion of an important role for cytosolic Ca(2+) signalling in jasmonate biosynthesis following OG perception. Promoter analysis of OG-induced, inhibitor-sensitive and -insensitive genes identified several putative cis-elements that might be involved specifically in Ca(2+)-dependent transcriptional regulation.

Immunochemical Analysis of Aquaporin Isoforms inArabidopsisSuspension-Cultured Cells

Aquaporins mediate the movement of water across biomembranes. Arabidopsis thaliana contains 35 aquaporins that belong to four subfamilies (PIP, TIP, SIP, and NIP). We investigated their expression profiles immunochemically in suspension-cultured Arabidopsis thaliana cells during growth and in response to salt and osmotic stresses. Protein amounts of all aquaporins were much lower in cultured cells than in the plant tissues. This is consistent with the low water permeability of protoplasts from cultured cells. After treatment with NaCl, the protein amounts of PIP2;1, PIP2;2, and PIP2;3 in the cells increased several-fold, and those of TIP1;1 and TIP1;2, 15- and 3-fold respectively. PIP1 did not change under the stress. Cell death began after 19 d in culture, accompanied by marked accumulation of PIPs and TIPs and a gradual decrease in SIPs. Our results suggest the followings: (i) Accumulation of aquaporin isoforms was individually regulated at low levels in single cells. (ii) At least PIP2;2, PIP2;3, TIP1;1, and TIP1;2 are stress-responsive aquaporins in suspension cells. (iii) A sudden increment of several members of PIP2 and TIP1 subfamilies might be related to cell death.

Loss of AtPIN1 does not influence the in vitro morphogenic potential of Arabidopsis thaliana suspension cultures

Suspension cultures from Arabidopsis thaliana wild type and AtPIN1-deficient lines were initiated and maintained for more than 3 years. A protocol for efficient regeneration from long-term suspension cultures was established. Arabidopsis wild-type and respectively AtPIN1 mutant plants have been regenerated from these cultures and characterized. Additionally, transgenic suspension cultures expressing the uidA (β -glucuronidase) reporter gene under the control of AtPIN1 promoter have been used for morphogenic studies. Our studies suggest that a lack of AtPIN1 function affects shoot differentiation and development, but does not influence in vitro regeneration of plants.

Physico-chemical characteristics of cell walls from Arabidopsis thaliana microcalli showing different adhesion strengths.

Changes in the composition and structure of cell walls and extracellular polysaccharides (ECP) were studied during the growth of suspension-cultured Arabidopsis thaliana microcalli. Three growth phases, namely the cell division phase, the cell expansion phase, and the stationary phase, were distinguished and associated with a decreasing cell cluster adhesion strength. Degradation of the homogalacturonan pectic backbone and of linear pectic side chains (1,4)-beta-D-galactan were observed concomitantly with the cell expansion and stationary phases and the decrease in cell adhesion. Also, in the stationary phase, branched (1,5)-alpha-L-arabinans were linearized. The AGP content of the culture medium increased while it decreased in the cell wall during cell growth and as cell adhesion decreased. These data suggest that, in addition to homogalacturonan, pectic side chains and AGP are involved in plant cell development and particularly in cell-cell attachment.

Hydrogen peroxide–induced gene expression in Arabidopsis thaliana

Hydrogen peroxide (H 2O 2) is generated in plants after exposure to a variety of biotic and abiotic stresses, and has been shown to induce a number of cellular responses. Previously, we showed that H 2O 2 generated during plant-elicitor interactions acts as a signaling molecule to induce the expression of defense genes and initiate programmed cell death in Arabidopsis thaliana suspension cultures. Here, we report for the first time the identification by RNA differential display of four genes whose expression is induced by H 2O 2. These include genes that have sequence homology to previously identified Arabidopsis genes encoding a late embryogenesis-abundant protein, a DNA-damage repair protein, and a serine/threonine kinase. Their putative roles in H 2O 2-induced defense responses are discussed.

Short communication. H2O2 activates a MAP kinase-like enzyme in Arabidopsis thaliana suspension cultures

Short communication. H2O2 activates a MAP kinase-like enzyme in Arabidopsis thaliana suspension cultures