Modification of plant Rac/Rop GTPase signalling using bacterial toxin transgenes.

Bacterial protein toxins which modify Rho GTPase are useful for the analysis of Rho signalling in animal cells, but these toxins cannot be taken up by plant cells. We demonstrate in vitro deamidation of Arabidopsis Rop4 by Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1) and glucosylation by Clostridium difficile toxin B. Expression of the catalytic domain of CNF1 caused modification and activation of co-expressed Arabidopsis Rop4 GTPase in tobacco leaves, resulting in hypersensitive-like cell death. By contrast, the catalytic domain of toxin B modified and inactivated co-expressed constitutively active Rop4, blocking the hypersensitive response caused by over-expression of active Rops. In transgenic Arabidopsis, both CNF1 and toxin B inhibited Rop-dependent polar morphogenesis of leaf epidermal cells. Toxin B expression also inhibited Rop-dependent morphogenesis of root hairs and trichome branching, and resulted in root meristem enlargement and dwarf growth. Our results show that CNF1 and toxin B transgenes are effective tools in Rop GTPase signalling studies.

Plant Growth Regulation in Cell and Tissue Culture In Vitro.

Precise knowledge of all aspects controlling plant tissue culture and in vitro plant regeneration is crucial for plant biotechnologists and their correlated industry, as there is increasing demand for this scientific knowledge, resulting in more productive and resilient plants in the field. However, the development and application of cell and tissue culture techniques are usually based on empirical studies, although some data-driven models are available. Overall, the success of plant tissue culture is dependent on several factors such as available nutrients, endogenous auxin synthesis, organic compounds, and environment conditions. In this review, the most important aspects are described one by one, with some practical recommendations based on basic research in plant physiology and sharing our practical experience from over 20 years of research in this field. The main aim is to help new plant biotechnologists and increase the impact of the plant tissue culture industry worldwide.

Different stresses, similar morphogenic responses: integrating a plethora of pathways.

Exposure of plants to mild chronic stress can cause induction of specific, stress-induced morphogenic responses (SIMRs). These responses are characterized by a blockage of cell division in the main meristematic tissues, an inhibition of elongation and a redirected outgrowth of lateral organs. Key elements in the ontogenesis of this phenotype appear to be stress-affected gradients of reactive oxygen species (ROS), antioxidants, auxin and ethylene. These gradients are present at the the organismal level, but are integrated on the cellular level, affecting cell division, cell elongation and/or cell differentiation. Our analysis of the literature indicates that stress-induced modulation of plant growth is mediated by a plethora of molecular interactions, whereby different environmental signals can trigger similar morphogenic responses. At least some of the molecular interactions that underlie morphogenic responses appear to be interchangeable. We speculate that this complexity can be viewed in terms of a thermodynamic model, in which not the specific pathway, but the achieved metabolic state is biologically conserved.

Stress-induced morphogenic responses: growing out of trouble?

Plants exposed to sub-lethal abiotic stress conditions exhibit a broad range of morphogenic responses. Despite the diversity of phenotypes, a generic 'stress-induced morphogenic response' can be recognized that appears to be carefully orchestrated and comprises three components: (a) inhibition of cell elongation, (b) localized stimulation of cell division and (c) alterations in cell differentiation status. It is hypothesized that the similarities in the morphogenic responses induced by distinct stresses, reflect common molecular processes such as increased ROS-production and altered phytohormone transport and/or metabolism. The stress-induced morphogenic response (SIMR) is postulated to be part of a general acclimation strategy, whereby plant growth is redirected to diminish stress exposure.

The involvement of reactive oxygen species (ROS) in the cell cycle activation (G(0)-to-G(1) 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 (G(0)-to-G(1)) and may have a positive effect on the plant cell cycle machinery. A generalized model for concentration-dependent synergistic effect of auxin and ROS on differentiated plant cells is also shown.

Nitric oxide is required for, and promotes auxin-mediated activation of, cell division and embryogenic cell formation but does not influence cell cycle progression in alfalfa cell cultures.

It is now well established that nitric oxide (NO) serves as a signaling molecule in plant cells. In this paper experimental data are presented which indicate that NO can stimulate the activation of cell division and embryogenic cell formation in leaf protoplast-derived cells of alfalfa in the presence of auxin. It was found that various NO-releasing compounds promoted auxin-dependent division (as shown by incorporation of bromodeoxyuridine) of leaf protoplast-derived alfalfa cells. In contrast, application of NO scavenger or NO synthesis inhibitor inhibited the same process. Both the promotion and the inhibition of cell cycle activation correlated with the amount and activity of the cognate alfalfa p34cdc2 protein Medsa;CDKA;1,2. The effect of l-NG-monomethyl-L-arginine (L-NMMA) was transient, and protoplast-derived cells spending more than 3 days in culture become insensitive to the inhibitor as far as cell cycle progression was concerned. L-NMMA had no effect on the cell cycle parameters of cycling suspension-cultured cells, but had a moderate transient inhibitory effect on cells re-entering the cell cycle following phosphate starvation. Cycling cultured cells, however, could respond to NO, as indicated by the sodium nitroprusside (SNP)- and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO)-dependent accumulation of the ferritin protein. Based on these observations, it is hypothesized that L-NMMA-sensitive generation of NO is involved in the activation, but not the progression of the plant cell division cycle. In addition, SNP promoted and L-NMMA delayed the exogenous auxin [2,4-dichlorophenoxyacetic acid (2,4-D)] concentration-dependent formation of embryogenic cell clusters expressing the MsSERK1 gene; this further supports a link between auxin- and NO-dependent signaling pathways in plant cells.

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 microM) or high (10 microM) 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 microM 2,4-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 microM 2,4-D-containing culture medium by 10 mM 2-(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.