Arabidopsis Thaliana Cell Suspension Cultures Research Articles

Effect of auxin, cytokinin, and sucrose on cell cycle gene expression in Arabidopsis thaliana cell suspension cultures

Cell suspension cultures of Arabidopsis thaliana (L.) Heynh. have been used to study the effect of auxin (α-naphthaleneacetic acid), cytokinin (kinetin), and sucrose on cell cycle gene expression. Auxin, cytokinin, and/or sucrose were re-added to culture media depleted for all three mitogens and the effects on the transcript levels of key cell cycle genes were monitored. Different cell cycle genes responded differentially to the mitogen supply. The cyclin-dependent kinase (CDK)-encoding gene CDKA;1 and the A- and D-type cyclins were induced by sucrose, whereas CDKB1;1 and CYCB1;1 were induced most strongly by the combined administration of auxin and cytokinin. The transcript levels of CDK inhibitors were up-regulated upon the addition of Gamborg's B5 salts to depleted medium. This induction was counteracted by the presence of auxin, cytokinin, or sucrose, depending on the CDK inhibitor. The obtained results are discussed in regard to the current knowledge on mitogen effects on cell cycle regulation in Arabidopsis.

Analysis of cell division parameters and cell cycle gene expression during the cultivation of Arabidopsis thaliana cell suspensions

Arabidopsis thaliana cell suspension cultures were characterized for the first time in detail in terms of biomass accumulation, cell division rate and cell cycle phase durations. Subsequently, this model system was used to follow the transcription profile of key cell cycle genes during a complete cultivation cycle. According to the calculated changes in the relative division rate over time, the cell cycle genes could be classified into four groups based on their transcriptional expression pattern. These differential patterns of gene expression are discussed with respect to the putative roles of the different cell cycle genes in the division cycle. Analysis of protein levels showed that mRNA levels did not correlate with protein levels in all cases. Results obtained in other systems, such as BY-2 cell suspensions or plants, confirm that cell suspension cultures of A. thaliana are suitable for the analysis of cell cycle regulation.

An improved protocol for the preparation of protoplasts from an established Arabidopsis thaliana cell suspension culture and infection with RNA of turnip yellow mosaic tymovirus: a simple and reliable method

An improved method for preparation of protoplasts of Arabidopsis thaliana cells grown in suspension culture is presented. This method is fast, reliable and can be used for the production of virtually an unlimited number of protoplasts at any time. These protoplasts can be transformed efficiently with RNA from turnip yellow mosaic tymovirus (TYMV) by polyethyleneglycol-mediated transfection. The simple transfection procedure has been optimized at various steps. Replication of TYMV can be monitored routinely by detection of the coat protein in as few as 2×10 4 infected protoplasts.

Salinity and Hyperosmotic Stress Induce Rapid Increases in Phosphatidylinositol 4,5-Bisphosphate, Diacylglycerol Pyrophosphate, and Phosphatidylcholine in Arabidopsis thaliana Cells

In animal cells, phosphoinositides are key components of the inositol 1,4,5-trisphosphate/diacylglycerol-based signaling pathway, but also have many other cellular functions. These lipids are also believed to fulfill similar functions in plant cells, although many details concerning the components of a plant phosphoinositide system, and their regulation are still missing. Only recently have the different phosphoinositide isomers been unambiguously identified in plant cells. Another problem that hinders the study of the function of phosphoinositides and their derivatives, as well as the regulation of their metabolism, in plant cells is the need for a homogenous, easily obtainable material, from which the extraction and purification of phospholipids is relatively easy and quantitatively reproducible. We present here a thorough characterization of the phospholipids purified from [(32)P]orthophosphate- and myo-[2-(3)H]inositol-radiolabeled Arabidopsis thaliana suspension-cultured cells. We then show that NaCl treatment induces dramatic increases in the levels of phosphatidylinositol 4,5-bisphosphate and diacylglycerol pyrophosphate and also affects the turnover of phosphatidylcholine. The increase in phosphatidylinositol 4,5-bisphosphate was also observed with a non-ionic hyperosmotic shock. In contrast, the increase in diacylglycerol pyrophosphate and the turnover of phosphatidylcholine were relatively specific to salt treatments as only minor changes in the metabolism of these two phospholipids were detected when the cells were treated with sorbitol instead of NaCl.

Direct measurement of ascorbic acid biosynthesis in Arabidopsis cell suspension culture using capillary electrophoresis

We describe procedures to directly measure the biosynthesis of vitamin C (l-ascorbic acid, l-AA) in crude extracts of an Arabidopsis thaliana cell suspension culture by capillary electrophoresis. Optimal conditions have been established for the quantitation of l-AA formed by the oxidation of three different substrates: l-galactose, l-galactono-1,4-lactone, and l-gulono-1,4-lactone. We also demonstrate that l-galactono-1,4-lactone dehydrogenase activity does not require exogenous cofactor. The minimal sample handling requirements, the high selectivity, and short analysis times represent significant advantages over existing protocols.

Yariv reagent treatment induces programmed cell death in Arabidopsis cell cultures and implicates arabinogalactan protein involvement.

Arabinogalactan proteins (AGPs) are a family of highly glycosylated, hydroxyproline-rich glycoproteins implicated in various aspects of plant growth and development. (beta-D-glucosyl)3 and (beta-D-galactosyl)3 Yariv phenylglycosides, commonly known as Yariv reagents, specifically bind AGPs in a non-covalent manner. Here (beta-D-galactosyl)3 Yariv reagent was added to Arabidopsis thaliana cell suspension cultures and determined to induce programmed cell death (PCD) by three criteria: (i) DNA fragmentation as detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) of DNA 3'-OH groups; (ii) inter- nucleosomal DNA fragmentation as visualized by genomic Southern blotting; and (iii) structural changes characteristic of PCD including cytoplasmic shrinkage and condensation, chromatin condensation and nuclear membrane blebbing. These findings implicate AGP involvement in PCD in plants, presumably by perturbation of AGPs located at the plasma membrane-cell wall interface.

Changes in functional properties of mitochondria during growth cycle of Arabidopsis thaliana cell suspension cultures

Composition and properties of Percoll-purified mitochondria isolated from Arabidopsis thaliana cell suspension cultures were studied at various stages of the growth cycle. During the lag phase (2 days) mitochondria exhibited high lipid/ protein ratio, high double bound index and very low oxidative rate. After day 2 and up to day 4 for NADH (exponential phase), or to day 7 (beginning of the senescence) for succinate as substrates, the oxidation rate in state 3 increased while oxidation rate in state 4 remained low. Consequently, mitochondria appeared more and more coupled over the whole time of the growth cycle. Along with the constant oxidation rate in state 4, characteristics of membranes remained similar in terms of polypeptide pattern, proton conductance or fluorescence anisotropy although unsaturation of membranous fatty acids increased significantly during the growth cycle. All these results indicated that oxidation rate in state 3 increased continuously while basic properties of mitochondrial membranes remained similar during the growth.

Identification of proliferation-induced genes in Arabidopsis thaliana. Characterization of a new member of the highly evolutionarily conserved histone H2A.F/Z variant subfamily.

The changes in gene expression associated with the reinitiation of cell division and subsequent progression through the cell cycle in Arabidopsis thaliana cell-suspension cultures were investigated. Partial synchronization of cells was achieved by a technique combining phosphate starvation and a transient treatment with the DNA replication inhibitor aphidicolin. Six cDNAs corresponding to genes highly induced in proliferating cells and showing cell-cycle-regulated expression were obtained by the mRNA differential display technique. Full-length cDNA clones (cH2BAt and cH2AvAt) corresponding to two of the display products were subsequently isolated. The cH2BAt clone codes for a novel histone H2B protein, whereas the cH2AvAt cDNA corresponds to a gene encoding a new member of the highly conserved histone H2A.F/Z subfamily of chromosomal proteins. Further studies indicated that H2AvAt mRNA expression is tightly correlated with cell proliferation in cell-suspension cultures, and that closely related analogs of the encoded protein exist in Arabidopsis. The implications of the conservation of histone H2A.F/Z variants in plants are discussed.

Structure and organ specificity of an anionic peroxidase from Arabidopsis thaliana cell suspension culture

The predominant peroxidase (pl 3.5) (E.C. 1.11.1.7) of an Arabidopsis thaliana cell suspension culture was purified and partially sequenced. Oligonucleotides were designed and a specific probe was obtained. A cDNA clone was isolated from an Arabidopsis cell suspension cDNA library and completely sequenced. The cDNA clone comprised 1194 bp and encodes a 30 residue signal peptide and a 305 residue mature protein ( M r 31 966). The sequence of the mature protein is 95% identical to the well-characterized horseradish peroxidase HRP A2 and has therefore been designated ATP A2. Three introns at positions identical to those found in Arabidopsis and horseradish genes encoding cationic peroxidases were identified. RT-PCR analysis revealed root-specific expression.

Olomoucine, an inhibitor of the cdc2/cdk2 kinases activity, blocks plant cells at the G1 to S and G2 to M cell cycle transitions

The cdc2/cdk2 protein kinases play key roles in the cell cycle at two control points: the G1/S transition and the entry into mitosis. Olomoucine, a specific inhibitor of these kinases, was tested in two plant cell systems: Petunia mesophyll protoplasts induced to divide and Arabidopsis thaliana cell suspension cultures. The cell cycle status was analysed from DNA histograms or through continuous labelling of cells with 5-bromodeoxyuridine (BrdUrd) followed by double staining with bis-benzimide (Hoechst 33258) and propidium iodide (PI). Such analyses resolve cells from several generations according to the extent of their DNA replication. Olomoucine was shown to reversibly arrest differentiated Petunia cells induced to divide at G1 phase and cycling Arabidopsis cells in late G1 and G2. A comparison of the effects of aphidicolin, oryzalin and olomoucine suggests that in the Arabidopsis cell suspension culture, a cdc2/cdk2-like kinase is activated at a restriction point in late G1.

Cloning and sequence analysis of a cDNA clone from Arabidopsis thaliana homologous to a proteasome α subunit from Drosophila

A cDNA clone isolated from an Arabidopsis thaliana cell suspension culture library showed considerable similarities to the proteasome 28 kDa α subunit of Drosophila [(1990) Gene 90, 235–241]. The 250 amino acid-long protein encoded by Arabidopsis TAS-g64 clone has important homologies in its primary structure and in the predicted secondary structure with the PROS-28.1 clone from Drosophila. The only divergence observed between the two sequences is for the 20 C-terminal amino acids. This subunit might share important functions in both kingdoms, as revealed by the important conservation between plants and animals. In plant cells it is encoded by a single-copy gene and probably regulated by stress and/or division.