The effect of mechanical stress (centrifugation) on the induction of nitric oxide (NO) formation and DNA fragmentation was investigated in leaf cells of Arabidopsis thaliana. Centrifuged and non-centrifuged leaves from wild-type and nitrate reductase (NR)nia1, nia2 double mutant, defective in the assimilation of nitrate, were labelled with 4,5-diaminofluorescein diacetate (DAF-2 DA) to visualize in vivo NO production. After these treatments, DNA fragmentation was detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end in situ labelling (TUNEL) method. Exposure to an NO-releasing compound, sodium nitroprusside (SNP) mimicked the cell response to centrifugation (20 g). The involvement of endogenous NO as a signal in mechanical stress and in DNA fragmentation was confirmed by inhibition of NO production using a nitric oxide synthase (NOS) inhibitor viz. NG-monomethyl- L -arginine (L -NMMA). These results indicate that NOS-like activity was present in A. thaliana leaves and was increased by mechanical stress. The effect of leaf-wounding on nitric oxide production was identical to that of centrifugation. Experiments with A. thaliana NR mutant also showed that NO bursts were induced by mechanical and wounding stresses and that NO was not a by-product of NR activity. A positive and significant correlation between NO production and DNA fragmentation was recorded for both centrifuged and non-centrifuged cells. Our results suggest that factors other than NO contribute to DNA damage and cell death, and furthermore, that an inducible form of NOS is present in A. thaliana.

Leaves and callus of Kalanchoë daigremontiana and Taxus brevifolia were used to investigate nitric oxide-induced apoptosis in plant cells. The effect of nitric oxide (NO) was studied by using a NO donor, sodium nitroprusside (SNP), a nitric oxide-synthase (NOS) inhibitor, N:(G)-monomethyl-L-arginine (NMMA), and centrifugation (an apoptosis-inducing treatment in these species). NO production was visualized in cells and tissues with a specific probe, diaminofluorescein diacetate (DAF-2 DA). DNA fragmentation was detected in situ by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) method. In both species, NO was detected diffused in the cytosol of epidermal cells and in chloroplasts of guard cells and leaf parenchyma cells. Centrifugation increased NO production, DNA fragmentation and subsequent cell death by apoptosis. SNP mimicked centrifugation results. NMMA significantly decreased NO production and apoptosis in both species. The inhibitory effect of NMMA on NO production suggests that a putative NOS is present in Kalanchoë and Taxus cells. The present results demonstrated the involvement of NO on DNA damage leading to cell death, and point to a potential role of NO as a signal molecule in these plants.

Plant cells either die by "accident" (traumatic cell death) or by "design" (programmed cell death; PCD). There is clear evidence that cell death during plant development and interactions with the environment involves PCD (in Gray and Johal, 1998). K. daigremontiana reproduces asexually by forming plantlets from leaf indentations which fall to soil and convert into adult plants. In nature, its entire plant body except leaf-plantlets senesces as consequence of floral differentiation or stressful environmental conditions. At unit gravity, PCD precedes plantlet detachment from the mother-leaf, leading to an abscission scar after plantlet fall. Earlier experiments have shown that leaf-plantlet formation and asexual reproduction increased with short duration hypergravity treatments and decreased in simulated hypergravity (Pedroso and Durzan, 1998). The present experiments were designed to determine if and what type of cell death occurs following gravitational changes, and the sequence of events leading to it. Our study shows that changes in gravitational environment cause a burst in nitric oxide, followed by a sequence of events that may ultimately led to programmed cell death by apoptosis.

Ten-year-old white spruce trees were evaluated for their ability to produce female cones in response to night-interrupted exposures of red light. Under field conditions, red-light treatments varied with time during the growing season, frequency of treatment within each cycle, and duration of exposure (dosage).

The ratio of basic to acidic amino acids in protein from free virions of CPV of Malacosoma disstria was 0.26, while that of occluded virions liberated from the polyhedra was 0.43. The ratio in free virions of CPV of Orgyia leucostigma was 0.39. This suggests that the virions of O. leucostigma reach a more advanced stage of maturity before occlusion than do those of M. disstria. By comparison, the composition of the free virions of O. leucostigma CPV was remarkably similar to that of the midgut protein of host cells. On the other hand, the composition of ribosomes of M. disstria was significantly different from either free or occluded virions of M. disstria CPV. Protein amino acids from virions of Bombyx mori differed from those of virions of other hosts in content of basic amino acids yielding a ratio of 0.68. These characteristics help to identify the viral strains and support previous serological studies reported with these proteins.

Levels of free and to a lesser extent bound amino acids in larvae of the spruce budworm (Choristoneura fumiferana (Clem.)) varied significantly with food source (host species) and date of sampling (instars V and VI). Amino acids most prominent and occurring free were glutamine, glutamic acid, alanine, and proline, whereas those bound and released by acid hydrolysis were mainly aspartic and glutamic acids. Arginine contributed most to the protein N of larvae and in the host species was the main storage form of nitrogen. Percentages of twelve free amino acids including ornithine, glutamic and γ-aminobutyric acid, lysine, valine, leucine, and isoleucine reflected significantly food sources, sampling dates, and their interactions. When feeding stopped (instar VI), levels of most bound amino acids in red spruce and fir larvae increased/unit weight as levels of free amino acids dropped. Although percentages of most bound amino acids were unaffected by diet, significant differences were found with glutamic acid, histidine, and threonine. Compared to white and red spruce, balsam fir, the preferred host in the sampled area, provided larvae with the highest levels of free and bound amino acids and greatest size.