Inheritance of the Reversal of O(2) Response of Photosynthesis in a Flaveria linearis Mutant

A mutant plant of Flaveria linearis Lag. expresses reversed O₂ response of photosynthesis (i.e. its apparent photosynthesis is stimulated at atmospheric O₂ levels). The objectives of this study were to determine the genetic inheritance of this trait and to investigate the biochemical mechanism for its expression. The mutant plant was crossed reciprocally with a plant of the closely related species Flaveria oppositifolia (DC.) Rydb. and also with another plant of F. linearis. Data on O₂ inhibition of apparent photosynthesis were analyzed on F₂ and F₃ progeny from these F₁ hybrids. In addition, test crosses (mutant × F₁ hybrid) and S₁ progeny from the mutant plant were also analyzed. All F₁ hybrids expressed inhibition of apparent photosynthesis and their progeny segregated in acceptable 3:1 and 13:3 (normal:reversed) ratios. There was little effect of environment on expression of the reversed O₂ response. Selected F₂ plants and the original mutant plant produced progeny in normal:reversed ratios which indicated the trait is controlled by two major genes which show dominant and recessive epistasis. Plants with greater than 20 nanomoles per gram fresh weight per minute of fructose-1, 6-bisphosphatase activity in the cytosol had normal O₂ response of photosynthesis. However, when plants had less than 20 nanomoles per gram fresh weight per minute of this enzyme activity in the cytosol, the O₂ was normal in some and reversed in others. It is proposed that low fructose bisphosphatase activity in the cytosol is controlled by a recessive gene (fbp). A second dominant gene is speculated to be hypostatic to the normal fructose bisphosphatase gene and controls the expression of an unknown factor that determines whether O₂ response of AP is reversed in the presence of fbp (i.e. when fructose bisphosphatase activity is low).     

Photodynamics of Porphyric Insecticides

The discovery of porphyric insecticides was a direct fallout of the discovery and development of photodynamic herbicides. Tetrapyrrole-dependent photodynamic herbicides are compounds that force green plants to accumulate undesirable amounts of metabolic intermediates of the chlorophyll and heme metabolic pathways, namely, tetrapyrroles. In light, the accumulated tetrapyrroles photosensitize the formation of singlet oxygen that kills treated plants by oxidation of their cellular membranes. Demonstration of the potential for tetrapyrrole accumulation in insects was achieved by spraying T. ni larvae with δ-aminolevulinic acid (ALA) and 2,2-dipyridyl (Dpy). Treated larvae were placed overnight in darkness at 28°C in order to allow for tetrapyrrole accumulation. Extraction of treated, dark-incubated larvae with ammoniacal acetone, followed by spectrofluorometric examination of the larval extract, revealed the accumulation of massive amounts of protoporphyrin IX (Proto). A high degree of correlation was observed between Proto accumulation in darkness and larval death in the light. A few hours after exposure to light, the larvae became sluggish and flaccid due to loss of body fluids. Death was accompanied by extensive desiccation. Because control of insects by ingestion is as viable an option as control by spraying, and offers certain advantages under household conditions, studies were conducted to determine whether combinations of ALA and porphyric insecticide modulators would be effective if ingested with the food. The effect of ALA and 1,10-phenanthroline (Oph) were determined by incorporating them into the diet of T. ni larvae. After exposure to light, following 17 h of dark incubation, larvae underwent violent convulsions and vomiting and died within 20 to 40 s. Tetrapyrrole analysis of the treated larvae immediately after dark incubation revealed significant amounts of Proto and Zn-Proto accumulation. Correlation between tetrapyrrole accumulation and larval death was significant. Similar results were obtained when ALA and Dpy were administered to the larvae with the diet. The above results indicated that in addition to contact via spraying, porphyric insecticides had the potential to be very potent when ingested. For a more thorough understanding of the mode of action of porphyric insecticides, the phenomenology of tissue, cellular, and subcellular sites of tetrapyrrole accumulation in representative insect species was investigated. In T. ni larvae, on a unit protein basis, about 59% of the accumulated Proto was observed in the hemolymph, 35% in the gut, and 6% in the integument. Further understanding of the response of insect organs and tissues to porphyric insecticide treatment was obtained by investigating the response of isolated organs and tissues to incubation with ALA + Dpy or ALA + Oph in adult Blattella germanica (German cockroach), adult Anthonomus grandis (cotton boll weevil), fifth instar larvae of Heliothus zea (corn earworm), and fifth instar larvae of T. ni (cabbage looper). In T. ni, and H. zea, significant Proto accumulation was observed in incubated midgut and fat bodies. Proto accumulation occurred when tissues were incubated with Dpy, ALA + Dpy, Oph, and ALA + Oph (2). No response to treatment with ALA alone was observed. In cockroaches, more of the Proto appeared to accumulate in the male and female guts than in their abdomen. As in T. ni and H. zea, the response was elicited by each of the treatments that included Dpy or Oph. Cotton boll weevil abdomens appeared to be less responsive than the abdomens of the other three species. To determine whether Proto accumulation resulted in photodynamic damage of incubated tissues, T. ni midguts were incubated in darkness either in buffer, with ALA, or with Oph + ALA. Oxygen consumption of the tissue was monitored before and after exposure to 2-h of illumination. A 30% decrease in O₂ consumption was observed in midguts treated with Oph or with ALA + Oph after 2 h in the light. The decrease in oxygen consumption observed in isolated T. ni midguts was shown to be caused by photodynamic damage to mitochondrial enzymes. Finally, structure-function photodynamic insecticidal studies led to the identification of 36 compounds belonging to 10 different chemical families that were effective (>70% mortality) against at least one insect species. Of the 36 modulators, 10 exhibited potent activity toward cockroaches.     

Oxygen Stimulation of Apparent Photosynthesis in Flaveria linearis

A plant was found in the C₃-C₄ intermediate species, Flaveria linearis, in which apparent photosynthesis is stimulated by atmospheric O₂ concentrations. A survey of 44 selfed progeny of the plant showed that the O₂ stimulation of apparent photosynthesis was passed on to the progeny. When leaves equilibrated at 210 milliliters per liter O₂ were transferred to 20 milliliters per liter O₂ apparent photosynthesis was initially stimulated, but gradually declined so that at 30 to 40 minutes the rate was only about 80 to 85% of that at 210 milliliters per liter O₂. Switching from 20 to 210 milliliters per liter caused the opposite transition in apparent photosynthesis. All other plants of F. linearis reached steady rates within 5 minutes after switching O₂ that were 20 to 24% lower in 210 than in 20 milliliters per liter O₂. At low intercellular CO₂ concentrations and low irradiances, O₂ inhibition of apparent photosynthesis of the aberrant plant was similar to that in normal plants, but at an irradiance of 2 millimoles quanta per square meter per second and near 300 microliters per liter CO₂ apparent photosynthesis was consistently higher at 210 than at 20 milliliters per liter O₂. In morphology and leaf anatomy, the aberrant plant is like the normal plants in F. linearis. The stimulation of apparent photosynthesis at air levels of O₂ in the aberrant plant is similar to other literature reports on observations with C₃ plants at high CO₂ concentrations, high irradiance and/or low temperatures, and may be related to limitation of photosynthesis by triose phosphate utilization.     

Photosynthesis of Grass Species Differing in Carbon Dioxide Fixation Pathways : VIII. Ultrastructural Characteristics of Panicum Species in the Laxa Group

Ultrastructural studies of leaves of seven Panicum species in or closely related to the Laxa group and classified as C₃, C₄ or C₃-C₄ intermediate were undertaken to examine features associated with C₃ and C₄ photosynthesis. The C₃ species Panicum rivulare Trin. had few organelles in bundle sheath cell profiles (2 chloroplasts, 1.1 mitochondria, and 0.3 peroxisomes per cell section) compared to an average of 10.6 chloroplasts, 17.7 mitochondria, and 3.2 peroxisomes per bundle sheath cell profile for three C₃-C₄ species, Panicum milioides Nees ex Trin., Panicum decipiens Nees ex Trin. and Panicum schenckii Hack. However, two other C₃ species, Panicum laxum Sw. and Panicum hylaeicum Mez, contained about 0.7, 0.5, and 0.3 as many chloroplasts, mitochondria, and peroxisomes, respectively, as in bundle sheath cell profiles of the C₃-C₄ species. Chloroplasts and mitochondria in bundle sheath cells were larger than those in mesophyll cells for the C₄ species Panicum prionitis Griseb. and the C₃-C₄ species, but in C₃ species the organelles were similar in size or were smaller in the bundle sheath cells. The C₃-C₄ species and P. laxum and P. hylaeicum exhibited an unusually close association of organelles in bundle sheath cells with mitochondria frequently surrounded in profile by chloroplasts. The high concentrations in bundle sheath cells of somewhat larger organelles than in mesophyll cells correlates with the reduced photorespiration of the C₃-C₄ species.     

A paradoxical pro-apoptotic effect of thrombin on smooth muscle cells

Whereas thrombin (below 10 nM) is a potent mitogen, recent studies report that exposure to higher doses of thrombin could lead to apoptosis of neurons and tumor cells. Our results show that prolonged exposure (> or = 24 h) to thrombin (50-100 nM) exerts a pro-apoptotic effect on cultured vascular smooth muscle cells (VSMCs). This phenomenon depends on thrombin serine-protease activity but is independent of PAR-1 and -4 activation and subsequent signaling. The parallel occurrence of cell retraction and cleavage of fibronectin suggests that thrombin-induced apoptosis is consecutive to pericellular proteolysis. These data point to a new potential action of thrombin in the cardiovascular system.     

Protease nexin-1 inhibits plasminogen activation-induced apoptosis of adherent cells

Degradation of adhesive glycoproteins by plasmin is implicated in cell migration. In this study, we further explored the role of plasminogen activation in cell adhesion and survival and show that uncontrolled plasminogen activation at the cell surface may induce cell detachment and apoptosis. We hypothesized that this process could be prevented in adherent cells by expression of protease nexin-1, a potent serpin able to inhibit thrombin, plasmin, and plasminogen activators. Using two- and three-dimensional culture systems, we demonstrate that Chinese hamster ovary fibroblasts constitutively express tissue-type plasminogen activator and efficiently activate exogenously added plasminogen in a specific and saturable manner (K(m) = 46 nm). The formation of plasmin results in proteolysis of fibronectin and laminin, which is followed by cell detachment and apoptosis. Protease nexin-1 expressed by transfected cells significantly inhibited the activity of plasmin and tissue-type plasminogen activator via the formation of inhibitory complexes and prevented cell detachment and apoptosis. In conclusion, protease nexin-1 may be an important anti-apoptotic factor for adherent cells. This cell model could be a useful tool to evaluate therapeutic agents such as serpins in vascular pathologies involving pericellular protease-protease inhibitor imbalance.