Limited ventilation causes stress and changes in Arabidopsis morphological, physiological and molecular phenotype during in vitro growth.

A huge number of experiments in plant biology are conducted in sterile, sealed containers, providing environmental stability and full control of factors influencing the plant system. With respect to roots the in vitro growth has another benefit - the ease of conducting visual observations when grown in transparent media. Moreover, straightforward measurements of in vitro grown root systems make them a sensitive and convenient sensor of multiple stresses which may occur during experiments. In order to optimize root nematode infection tests for Arabidopsis mutants with relatively mild phenotypes, two Petri dish sealing techniques were tested (permeable medical adhesive tape and a popular non-permeable plastic film). Using standard experimental settings applied for infection tests, the root architecture, nematode infections, ion leakage, efficiency of photosynthesis, ethylene (ET) production, and CO2 accumulation were monitored in Arabidopsis thaliana Ws-0 wild-type and lsd1 (lesion stimulating disease 1) plants, which is a conditional dependent programmed cell death mutant. All tested parameters gave statistically significant differences between the analyzed sealing tapes, indicating the importance of air exchange. This factor is quite obvious but often ignored in experiments performed in Petri dishes. The results clearly indicate that stress is greater in air-tight sealed plates. These observations were supported by the great expression variation of several marker genes associated with reactive oxygen species (ROS), ET, salicylic (SA), and jasmonic acid (JA) biosynthesis and signaling in two-week-old seedlings. These results are discussed in light of the observed changes in the ET and CO2 concentration. Our results clearly indicate the importance of culture parameters for monitoring of abiotic and biotic stress responses in laboratory conditions, including accurate mutant phenotyping.

Cross-talk between high light stress and plant defence to the two-spotted spider mite in Arabidopsis thaliana.

Little is known about how plants deal with arthropod herbivores under the fluctuating light intensity and spectra which occur in natural environments. Moreover, the role of simultaneous stress such as excess light (EL) in the regulation of plant responses to herbivores is poorly characterized. In the current study, we focused on a mite-herbivore, specifically, the two-spotted spider mite (TSSM), which is one of the major agricultural pests worldwide. Our results showed that TSSM-induced leaf damage (visualized by trypan blue staining) and oviposition rate (measured as daily female fecundity) decreased after EL pre-treatment in wild-type Arabidopsis plants, but the observed responses were not wavelength specific. Thus, we established that EL pre-treatment reduced Arabidopsis susceptibility to TSSM infestation. Due to the fact that a portion of EL energy is dissipated by plants as heat in the mechanism known as non-photochemical quenching (NPQ) of chlorophyll fluorescence, we tested an Arabidopsis npq4-1 mutant impaired in NPQ. We showed that npq4-1 plants are significantly less susceptible to TSSM feeding activity, and this result was not dependent on light pre-treatment. Therefore, our findings strongly support the role of light in plant defence against TSSM, pointing to a key role for a photo-protective mechanism such as NPQ in this regulation. We hypothesize that plants impaired in NPQ are constantly primed to mite attack, as this seems to be a universal evolutionarily conserved mechanism for herbivores.

Analysis of tomato gene promoters activated in syncytia induced in tomato and potato hairy roots by Globodera rostochiensis.

The potato cyst nematode (Globodera rostochiensis) induces feeding sites (syncytia) in tomato and potato roots. In a previous study, 135 tomato genes up-regulated during G. rostochiensis migration and syncytium development were identified. Five genes (CYP97A29, DFR, FLS, NIK and PMEI) were chosen for further study to examine their roles in plant-nematode interactions. The promoters of these genes were isolated and potential cis regulatory elements in their sequences were characterized using bioinformatics tools. Promoter fusions with the ß-glucuronidase gene were constructed and introduced into tomato and potato genomes via transformation with Agrobacterium rhizogenes to produce hairy roots. The analysed promoters displayed different activity patterns in nematode-infected and uninfected transgenic hairy roots.

Induction of apoptosis and NF-kappaB by quercetin in growing murine L1210 lymphocytic leukaemic cells potentiated by TNF-alpha.

Polyphenol quercetin induced apoptosis in proliferating murine L1210 lymphocytic cells. DNA damage, as well as apoptosis and withdrawal from the cell cycle were transient. The above mentioned death promoting activity of quercetin was enhanced by physiological concentrations of TNF-alpha. At the same time, indices of cell viability dropped. However, the extent and tendency of the initially enhanced cell mortality steadily diminished throughout the experiment. After 12 h the G2/M phase reappeared. After 24 h all indices almost returned to control levels indicating either the selection of subpopulation of unaffected leukaemic cells or cells developing resistance to the treatment. A DNA ladder of oligonucleosomes was observed for apoptogenic treatments. We conclude that quercetin unmasked cell death, promoting the activity of TNF-alpha. However, after 12 and 24 h of exposure, surviving cells could complete the cell cycle and finally recover. At the same time, increased NF-kappaB activation was demonstrated by immunoblotting of the immunoreactive RelA/p65 subunit in nuclear extracts. Exposure to TNF-alpha or quercetin was crucial for increased activity of NF-kappaB, which may implicate an increasing resistance to their cytotoxicity.

Apoptosis and Bcl-2 protein changes in L1210 leukaemic cells exposed to oxidative stress.

The aim of this study was to explore the dose- and time-dependent effects of hydrophilic peroxyl radical initiator 2,2'azobis(2amidinopropane)dihydrochloride (AAPH) on apoptosis, and on expression of Bcl-2 in L1210 leukaemic cells. We observed a progressive increase of intracellular concentration of oxygen free radicals (OFR), manifested by the rise of 6-carboxy-2', 7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) oxidation, during 24 h of cells exposure to AAPH. Oxidative stress was associated with peroxidation of cellular lipids, which was demonstrated by the measurement of thiobarbituric acid-reactive substances and conjugated dienes. Analysis of cell viability by the use of trypan blue exclusion method revealed that AAPH reduced the ability of L1210 cells to multiply or survive. AAPH increased the number of leukaemic cells with typical features of apoptosis like condensation of chromatin, pyknosis and fragmentation of nucleus, followed by secondary necrosis. A characteristic internucleosomal DNA cleavage, visualized as a DNA 'ladder' consisting of fragments that are multiples of 180-200 bp was also observed. The intensity of apoptosis was dependent on AAPH concentration, time of cell exposure and the availability of growth factors and nutrients in extracellular environment (FCS concentration). The novel observation is the increase of Bcl-2 level in L1210 leukaemic cells surviving an oxidative stress. The level of Bcl-2 protein significantly rose with increasing AAPH concentration, and time of cell exposure to this oxidant. This phenomenon could be the result of: (1) negative selection of cells with the lowest expression of bcl-2, being more susceptible to oxidative stress and (2) increased synthesis and/or decreased degradation of Bcl-2 protein as an adaptation to continuous OFR loading. In contrast to growth-promoting medium (10% FCS/RPMI), the maintenance medium (2% FCS/RPMI) did not cover cell requirements for progressive Bcl-2 increase at the highest AAPH concentration (2 mM) applied in this study. Several observations indicate that the increased Bcl-2 level in surviving L1210 leukaemic cells exposed to oxidative stress is a symptom of their natural defence against cellular lipids peroxidation and apoptosis.

Phorbol ester (12-O-tetradecanoylphorbol 13-acetate) prevents ornithine decarboxylase inhibition and apoptosis and L1210 leukemic cells exposed to TGF-beta 1.

Previous studies have shown that growth suppression and apoptosis of leukemic cells exposed to TGF-beta 1 is associated with the inhibition of ornithine decarboxylase (ODC)--the key enzyme of polyamine pathway. The aim of the present study was to evaluate the influence of 12-O-tetradecanoylphorbol 13-acetate (TPA)--a potent ODC inducer on antiproliferative and apoptotic effects of TGF-beta 1 in L1210 leukemic cells. Cells were incubated in 2% FCS/RPMI-1640 medium, supplemented with TGF-beta 1 (2 ng/ml). TPA (100 ng/ml) or alpha-difluoromethylornithine (DFMO) (5 mM). Cell proliferation, apoptosis and necrosis were evaluated using [methyl-3H] thymidine, electron microscopy, electrophoresis of DNA and trypan blue exclusion. Expression and activity of ODC were determined by RT-PCR and measurement of 14CO2 release from L-1-14C ornithine, respectively. TGF-beta 1 inhibited proliferation and induced apoptotic and necrotic cell death in L1210 leukemic cells. The above effects were associated with the inhibition of ODC expression and activity, measured 2 and 4 hr after TGF-beta 1 administration, respectively. The presence of DFMO, an irreversible inhibitor of ODC, led to apoptotic fragmentation of DNA, similar to that observed in TGF-beta 1-treated cultures. Administration of TPA simultaneously with TGF-beta 1 significantly reduced antiproliferative, apoptotic and necrotic effects of TGF-beta 1, and prevented its inhibitory action of ODC expression and activity. It is concluded that: down-regulation of ODC expression may be one of the early events associated with TGF-beta 1-evoked suppression of growth and apoptosis; ODC is involved in the mechanism of protective action of TPA on TGF-beta 1-related growth inhibition of L1210 leukemic cells.

Effect of orotic acid on TGF-beta 1-induced growth inhibition of L1210 leukemic cells.

UNLABELLED: Transforming growth factor-beta 1 (TGF-beta 1) exerted growth-inhibitory effect on L1210 leukemic cell line, manifested by the decrease in viable and increase in dead cells. The cell death evoked by TGF-beta 1 was both necrotic and apoptotic, quantified by the trypan blue exclusion method and apoptotic index, respectively. The induction of programmed cell death by TGF-beta 1 was confirmed by gel electrophoresis of DNA, where the characteristic 'DNA ladder' resulting from the internucleosomal DNA cleavage was visualized. The enhancement of cell mortality by TGF-beta 1 was associated with the inhibition of ornithine decarboxylase (ODC) expression (measured by the reverse transcriptase-polymerase chain reaction method) and impaired activity of this key enzyme in polyamine synthesis. Orotic acid (OA)--a known tumor promoter--stimulated proliferation of L1210 leukemic cells and diminished the necrotic effect of TGF-beta 1, but it did not change the extent of apoptosis evoked by TGF-beta 1. OA increased the expression of ODC and diminished depressional influence of TGF-beta 1 on transcription and activity of ODC in leukemic cells. IN CONCLUSION: OA is a bioactive compound stimulating the growth of leukemic cells and diminishing the growth-inhibitory effect of TGF-beta 1. ODC gene is probably one of the targets for both OA and TGF-beta 1 influences in L1210 leukemic cells.