Taras P. Pasternak

Transition of somatic plant cells to an embryogenic state

Under appropriate in vivo or in vitro conditions, certain somatic plant cells have the capability to initiate embryogenic development (somatic embryogenesis). Somatic embryogenesis provides an unique experimental model to understand the molecular and cellular bases of developmental plasticity in plants. In the last few years, the application of modern experimental techniques, as well as the characterization of Arabidopsis embryogenesis mutants, have resulted in the accumulation of novel data about the acquisition of embryogenic capabilities by somatic plant cells. In this review, we summarize relevant experimental observations that can contribute to the description and definition of a transitional state of somatic cells induced to form totipotent, embryogenic cells. During this somatic-to-embryogenic transition, cells have to dedifferentiate, activate their cell division cycle and reorganize their physiology, metabolism and gene expression patterns. The roles of stress, endogenous growth regulators and chromatin remodelling in the coordinated reorganization of the cellular state are especially emphasized.

The Role of Auxin, pH, and Stress in the Activation of Embryogenic Cell Division in Leaf Protoplast-Derived Cells of Alfalfa

Culturing leaf protoplast-derived cells of the embryogenic alfalfa (Medicago sativa subsp. varia A2) genotype in the presence of low (1 μm) or high (10 μm) 2, 4-dichlorophenoxyacetic acid (2,4-D) concentrations results in different cell types. Cells exposed to high 2,4-D concentration remain small with dense cytoplasm and can develop into proembryogenic cell clusters, whereas protoplasts cultured at low auxin concentration elongate and subsequently die or form undifferentiated cell colonies. Fe stress applied at nonlethal concentrations (1 mm) in the presence of 1 μm2,4-D also resulted in the development of the embryogenic cell type. Although cytoplasmic alkalinization was detected during cell activation of both types, embryogenic cells could be characterized by earlier cell division, a more alkalic vacuolar pH, and nonfunctional chloroplasts as compared with the elongated, nonembryogenic cells. Buffering of the 10 μm 2,4-D-containing culture medium by 10 mm2-(N-morpholino)ethanesulfonic acid delayed cell division and resulted in nonembryogenic cell-type formation. The level of endogenous indoleacetic acid (IAA) increased transiently in all protoplast cultures during the first 4 to 5 d, but an earlier peak of IAA accumulation correlated with the earlier activation of the division cycle in embryogenic-type cells. However, this IAA peak could also be delayed by buffering of the medium pH by 2-(N-morpholino)ethanesulfonic acid. Based on the above data, we propose the involvement of stress responses, endogenous auxin synthesis, and the establishment of cellular pH gradients in the formation of the embryogenic cell type.

Phytoglobins can interfere with nitric oxide functions during plant growth and pathogenic responses: a transgenic approach

To investigate the possible role of the non-symbiotic plant hemoglobins (phytoglobins) in relation to nitric oxide (NO) functions and their presumable involvement in NO- or pathogenesis-induced necrosis, we have produced transgenic tobacco plants (HOT lines) overexpressing an alfalfa hemoglobin cDNA (Mhb1 ) under the control of CaMV35S promoter. Upon treatment with active sodium nitroprusside (SNP), a widely used NO donor, the germination of seeds and development of seedlings were significantly less retarded in transgenic lines compared with the retardation of non-transformed seedlings. SNP-injection necrotized mature plant leaves of Mhb1 -transformants to a lower extent than control leaves. Furthermore, infection of tobacco leaves either with Pseudomonas syringae pv. phaseolicola or Tobacco Necrosis Virus (TNV) resulted in reduced necrosis of mature transgenic plants. In response to bacterial infection, reactive oxygen species (ROS) and salicylic acid (SA) were produced at a higher level in transgenic HOT plants than in control ones. The presented experimental data support a conclusion that plant non-symbiotic hemoglobins are active functional partners in NO-dependent physiological responses such as alteration of plant growth and development as well as cell death and symptom generation after pathogen infection. The described experiments provide new insights to the role of phytoglobins in ROS-, NO- and SA-mediated cellular events during the induction of necrotic cell death. # 2003 Elsevier Ireland Ltd. All rights reserved.

The involvement of reactive oxygen species (ROS) in the cell cycle activation (G0-to-G1 transition) of plant cells

Reactive oxygen species (ROS) are involved in various cellular processes in plants. Among those, resistance to abiotic stress, defence mechanisms and cell expansion have been intensively studied during the last years. We recently demonstrated that ROS, in concert with auxin, have a role in cell cycle activation of differentiated leaf cells.1 In this addendum we provide further evidence to show that oxidative stress/ROS accelerate auxin-mediated cell cycle entry (G0-to-G1 Addendum to: Pasternak TP, Ötvös K, Domoki M, Fehér A. Linked activation of cell division and oxidative stress defense in alfalfa leaf protoplast-derived cells is dependent on exogenous auxin. Plant Growth Regul 2007; 51:109-17.

Linked activation of cell division and oxidative stress defense in alfalfa leaf protoplast-derived cells is dependent on exogenous auxin

The activation of cell division and oxidative stress responses has been investigated in the case of leaf protoplast-derived cells. Initiation of protoplast culture was found to be associated with oxidative stress as indicated by the rate of H2O2 release into the medium and/or by catalase and ascorbate peroxidase activities. Both cell division frequency and the above stress-related parameters were dependent on the exogenous auxin (2,4-dichlorophenoxyacetic acid, 2,4-D) concentrations used. In addition, the well known oxidative stress-inducing agent paraquat (1xa0μM) could promote cell division at suboptimal auxin concentration but not in the absence of exogenous auxin. The H2O2 scavenger dimethylthiourea and the NADPH oxidase inhibitor diphenyleneiodonium inhibited not only the activation of cellular defense reactions but cell division as well. Based on the above experimental observations, it is suggested that exogenous auxin (2,4-D) enhances cellular defense reactions in parallel with cell division activation.

Exogenous auxin and cytokinin dependent activation of CDKs and cell division in leaf protoplast-derived cells of alfalfa

Alfalfa leaf protoplast cultures were used to study the role ofexogenously supplied auxin and cytokinin on the level and activity ofCdc2-related protein kinases and progression through the first celldivision cycle after re-activation of cell division. Among the threealfalfa Cdc2-related kinases studied, the Cdc2MsA/B kinase (PSTAIRE)showed only significant activity during the first four days ofprotoplast culture while the Cdc2MsD (PPTALRE) and Cdc2MsF kinases(PPTTLRE) exhibited only low or undetectable activity, respectively,during this period. Although the Cdc2MsA/B protein could be detectedin leaves and freshly isolated protoplasts in variable amounts, thekinase was never active in these cells. The kinase protein disappearedfrom protoplast-derived cells at the beginning (8h) of culture but itssynthesis re-commenced dependent on the presence of exogenous auxin butnot cytokinin. The cytokinin response of alfalfa protoplast-derivedcells varied significantly in different experiments although cytokininwas always required for completion of the first cell division cycle.Frequently both auxin and cytokinin was required for DNA replication asnot more than 5% of cells could incorporate BrdU into their DNAduring three days and significant Cdc2MsA/B activity could not bedetected in the absence of exogenous cytokinin. In other protoplastpopulations, the Cdc2MsA/B kinase was activated by auxin alone andallowed the protoplast-derived cells to enther the S-phase at a similarrate observed in parallel cultures with both auxin and cytokinin. Evenin these cultures, however, ca. 95% of the protoplast-derivedcells were arrested before mitosis without exogenous cytokinin supplywhich could be correlated with decreasing Cdc2MsA/B activity. Theseobservations suggest, that although cytokinin is required for bothG0-G1/S and G2/M cell cycle transitions, in certain cultures theG1/S requirement is overcome by some unknown factors (e.g.conditions of explants; endogenous cytokinins etc.). Furthermore, ourexperiments indicate, that the roles of cytokinin are related to thepost-translational regulation of the Cdc2MsA/B kinase complex atboth cell cycle transition points in alfalfa leaf protoplast-derivedcells. Finally, as a marker for the transition from the differentiated(G0) stage to the activated (G1) stage, we suggest using the parametersof nuclear morphology (size and ratio ofnucleus/nucleolus).

Cell-cycle, phase-specific activation of Maize streak virus promoters.

It is believed that geminiviral DNA replication is coupled to the cell-cycle regulatory complex of the plant cell and that the virus-early (complementary or C sense) gene products REP and REPA may be able to manipulate the regulation of the cycle. In this study, we examined expression from the promoters of Maize streak virus (MSV) in transgenic maize plants and cells to determine whether they showed cell-cycle specificity. Histochemical staining of plant roots containing long and short C-sense promoter sequences upstream of the GUS (beta-glucuronidase) reporter gene showed that promoter activity was restricted to the meristematic region of the roots and was enhanced by 2,4-dichlorophenoxy acetic acid (2,4-D) treatment. Analysis of reporter gene and cell-cycle-specific gene transcript levels coupled with flow cytometric data in synchronized transgenic maize cells revealed that all of the MSV promoters showed cell-cycle specificity. The coat protein gene promoter showed highest activity in early G2, whereas the C-sense promoter sequences produced two peaks of activity in the S and G2 cell-cycle phases.

Nitric oxide, a signaling molecule in plant cell reactivation

Nitric oxide is known to act as a biological messenger in divers signal transduction pathways in animal organisms. Initial investigations suggest that plants use nitric oxide as a signaling molecule via pathways remarkably similar to those found in mammals. Especially, the siganiling role of NO during plant defense reactions is well established. However, mounting evidences support the hypothesis that NO is a more general effector of plant growth and development. n nIn our laboratory, alfalfa cell cultures were used to investigate the possible involvement of NO in the regulation of cell division and differentiation in plant cells. The homogenous population of leaf protoplasts were cultured in the presence of a NO donor, sodium nitroprusside (SNP) and/or an inhibitor, NG-monomethyl-L-arginine (L-NMMA). BrdU incorporation frequency into the nuclei of the protoplast-derived cells indicated that the entry into the S-phase of the cell cycle is enhanced by SNP and inhibited by L-NMMA treatments, respectively (see Figure u200bFigure1).1). Experiments have also been carried out with continuously dividing cell suspension cultures. The obtained data indicated that these type of cells are insensitive to similar treatments shown to affect protoplast-derived cell division (see Figure u200bFigure22). n n n nFigure 1 n nBrdU incorporation frequency into the nuclei of leaf protplast-derived alfalfa cells during the third day of culture. The cells were treated by the indicated drugs affecting endogenous NO formation. For cell culture details see [2]. n n n n n nFigure 2 n nBrdU incorporation frequency into the nuclei of cell suspension-cultured alfalfa cells during the third day after subculture. The cells were treated by the indicated drugs affecting endogenous NO formation. For cell culture details see [2]. n n n nIn addition to cell cycle progression, the effect of the above drugs on the auxin-induced formation of embryogenic competent cells from leaf protoplasts (for more details see [1,2]) have been followed. The promotive effect of SNP and the inhibitory effect of L-NMMA have been observed on the process, especially at low exogenous auxin (0.02 μM 2,4-dichlorophenoxyacetic acid) supplementation.