The occurrence of crassulacean acid metabolism in Cymbidium (Orchidaceae) and its ecological and evolutionary implications.

Crassulacean acid metabolism (CAM) is one of the photosynthetic pathways regarded as adaptations to water stress in land plants. Little is known about correlations among the level of CAM activity, environment of habitat, life form, and phylogenetic relationship of a plant group from an evolutionary perspective. We examined these relationships in 18 species of Cymbidium (Orchidaceae) because the genus shows distinctive diversification of habitats and life forms. The photosynthetic type was classed into three categories, strong CAM, weak CAM, and C(3) on the basis of CAM activity. CAM expression in Cymbidium was confined to the epiphytic and lithophytic species. Especially, all of these species from tropical to subtropical rainforest exhibited CAM activity. On the other hand, the terrestrial species always exhibited C(3) metabolism irrespective of their varied habitats. Regarding the evolution of photosynthetic characters, weak CAM was the ancestral state in Cymbidium and strong CAM and C(3) metabolism occurred subsequently. The evolution of strong CAM likely enabled Cymbidium to extend to exposed sites in tropical lowland where marked water stress exists. Further, different levels of CAM activity characterized each species and such potential plasticity of CAM may realize the radiation of Cymbidium into sites with different environmental conditions.

Comparison of leaf structure and photosynthetic characteristics of C3 and C4 Alloteropsis semialata subspecies.

Alloteropsis semialata (R. Br.) Hitchcock includes both C3 and C4 subspecies: the C3 subspecies eckloniana and the C4 subspecies semialata. We examined the leaf structural and photosynthetic characteristics of these plants. A. semialata ssp. semialata showed high activities of photosynthetic enzymes involved in phosphoenolpyruvate carboxykinase-type C4 photosynthesis and an anomalous Kranz anatomy. Phosphoenolpyruvate carboxylase; pyruvate, Pi dikinase and glycine decarboxylase (GDC) were compartmentalized between the mesophyll (M) and inner bundle sheath cells, whereas ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) occurred in both cells. A. semialata ssp. eckloniana also showed an anomalous non-Kranz anatomy, in which the mestome sheath cells included abundant chloroplasts and mitochondria. Rubisco and GDC accumulated densely in the M and mestome sheath cells, whereas the levels of C4 enzymes were low. The activity levels of photo-respiratory enzymes in both subspecies were intermediate between those in typical C3 and C4 plants. The values of CO2 compensation points in A. semialata ssp. semialata were within the C4 range, whereas those in A. semialata ssp. eckloniana were somewhat lower than the C3 range. These data suggest that the plants are C3-like and C4-like but not typical C3 and C4, and when integrated with previous findings, point to important variability in the expression of C4 physiology in this species complex. A. semialata is therefore an intriguing grass species with which to study the evolutionary linkage between C3 and C4 plants.

Evidence from photosynthetic characteristics for the hybrid origin of Diplotaxis muralis from a C3-C4 intermediate and a C3 species.

Artificial hybridization studies have been carried out between plants with different photosynthetic types to study the genetic mechanism of photosynthetic types. However, there are only few reports describing the possibility of natural hybridization between plants with different photosynthetic types. A previous cytological and morphological study suggested that a cruciferous allotetraploid species, Diplotaxis muralis (L.) DC. (2n = 42), originated from natural hybridization between D. tenuifolia (L.) DC. (2n = 22) and D. viminea (L.) DC. (2n = 20). These putative parents have recently been reported to be a C (3)-C (4) intermediate and a C (3) species, respectively. If this hybridization occurred, D. muralis should have characteristics intermediate between those of the C (3)-C (4) intermediate and C (3) types. We compared leaf structures and photosynthetic characteristics of the three species. The bundle sheath (BS) cells in D. tenuifolia included many centripetally located chloroplasts and mitochondria, but those of D. viminea had only a few organelles. The BS cells in D. muralis displayed intermediate features between the putative parents. Glycine decarboxylase P protein was confined to the BS mitochondria in D. tenuifolia, but accumulated mainly in the mesophyll mitochondria in D. viminea. In D. muralis, it accumulated in both the BS and the mesophyll mitochondria. Values of CO (2) compensation point and its response to changing light intensity were also intermediate between the putative parents. These data support the theory that D. muralis was created by natural hybridization between species with different photosynthetic types.

Effect of light intensity on linear shrinkage of photo-activated composite resins during setting.

The purpose of this investigation was to examine the effects of light intensity on linear shrinkage of photo-activated composite resins during setting. The materials used were four commercially available photo-activated composite resins. Three light-irradiation instruments were selected and prepared so as to obtain four light intensities (200, 480, 800 and 1600 mW cm(-2)). The linear shrinkage during setting was examined 10 min after light irradiation using a trial balance plastometer, and the specimen thickness was 2.0 mm for all materials. The depth of cure was examined according to the test method described in the International Organization for Standardization (ISO/FDIS 4049: 2000(E)). In measuring the linear shrinkage 60 s from the start of light irradiation for 10 s, there was a significant correlation (r = 0.89-0.94) between the amount of linear shrinkage and the light intensity: an increase in light intensity produced a greater linear shrinkage. Furthermore, there was a significant correlation (r = 0.92-1.0) between the linear shrinkage and the irradiation time: an increase in irradiation time resulted in a greater linear shrinkage. Values of the depth of cure ranged from 1.69 to 3.75 mm.

Ultrastructural localization of photosynthetic and photorespiratory enzymes in epidermal, mesophyll, bundle sheath, and vascular bundle cells of the C4 dicot Amaranthus viridis.

In the leaves of the NAD-malic enzyme (NAD-ME)-type C4 dicot Amaranthus viridis L., there are chloroplasts in the vascular parenchyma cells (VPC), companion cells (CC), ordinary epidermal cells (EC), and guard cells (GC), as well as in the mesophyll cells (MC) and the bundle sheath cells (BSC). However, the chloroplasts of the VPC, CC, EC, and GC are smaller than those of the MC and BSC. In this study, the accumulation of photosynthetic and photorespiratory enzymes in these leaf cell types was investigated by immunogold labelling and electron microscopy. Strong labelling for phosphoenolpyruvate carboxylase was found in the MC cytosol. Weak labelling was observed in the CC and GC cytosol. Labelling for pyruvate, Pi dikinase occurred to varying degrees in the chloroplasts of all cell types except CC. Labelling for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase was detected in the chloroplasts of all cell types except MC. For both NAD-ME and the P-protein of glycine decarboxylase, intense labelling was found in the BSC mitochondria; weaker labelling was recognized in the VPC mitochondria. These data indicate that when not only the MC and BSC but also the other leaf cell types are included, the cell-specific expression of the enzymes in C4 leaves becomes more complex than has been known previously. These findings are discussed in relation to the metabolic function of epidermal and vascular bundle cells.

Species variation in the intracellular localization of pyruvate, Pi dikinase in leaves of crassulacean-acid-metabolism plants: an immunogold electron-microscope study.

In malic enzyme-dependent crassulacean-acid-metabolism (ME-CAM) plants, malic acid is decarboxylated by NADP-ME and NAD-ME and generates pyruvate with CO2. Pyruvate is phosphorylated to phosphoenolpyruvate by pyruvate, Pi dikinase (PPDK) and is then conserved in gluconeogenesis. Although PPDK was considered to be located in chloroplasts (e.g., Mesembryanthemum crystallinum), it has recently been found to accumulate in both the chloroplasts and the cytosol in two Kalanchoë species. In this study, the intracellular localization of PPDK was investigated in 22 ME-CAM species in 13 genera of 5 families by immunogold labeling and electron microscopy. This revealed that the pattern of intracellular localization of PPDK varies among the ME-CAM plants and is divided into three types: Chlt, in which PPDK accumulates only in the chloroplasts; Cyt-Chlt, in which PPDK accumulates in both chloroplasts and cytosol; and Cyt, in which PPDK accumulates predominantly in the cytosol. Members of a particular genus tend to have a common PPDK-localization type. In the Cactaceae, all species from seven genera were classified as Cyt. The photosynthetic tissues of all ME-CAM species, including the Cyt type, had substantial PPDK activity, suggesting that PPDK in the cytosol is active and probably plays a functional role. In the Chlt species, NADP-ME activity was relatively greater than NAD-ME activity. In the Cyt-Chlt and Cyt species, however, either the activity of NAD-ME was higher than that of NADP-ME or they were approximately the same. The species variation in the intracellular localization of PPDK is discussed in relation to CAM function and to molecular and phylogenetic aspects.

Localization of farnesyl diphosphate synthase in chloroplasts.

The subcellular localization of plant farnesyl diphosphate synthase (FPPS) was examined. Immunocytochemical staining using anti-FPPS1 antibody followed by electron microscopy showed that FPPS1 was localized to chloroplasts of rice mesophyll cells. Subcellular fractions from wheat leaves were examined by immunoblot analysis. FPPS was detected in the chloroplast fraction in wheat, and was protected from proteolysis following trypsin treatment of chloroplasts. FPPS was also detected in the chloroplast fraction of a dicot plant, tobacco.

Enzymes related to catecholamine biosynthesis in Tetrahymena pyriformis. Presence of GTP cyclohydrolase I.

We first identified GTP cyclohydrolase I activity (EC 3.5.4.16) in the ciliated protozoa, Tetrahymena pyriformis. The Vmax value of the enzyme in the cellular extract of T. pyriformis was 255 pmol mg-1 protein h-1. Michaelis-Menten kinetics indicated a positive cooperative binding of GTP to the enzyme. The GTP concentration producing half-maximal velocity was 0.8 mM. By high-performance liquid chromatography (HPLC) with fluorescence detection, a major peak corresponding to D-monapterin (2-amino-4-hydroxy-6-[(1'R,2'R)-1',2',3'-trihydroxypropyl]pteridin e, D-threo-neopterin) and minor peaks of D-erythro-neopterin and L-erythro-biopterin were found to be present in the cellular extract of Tetrahymena. Thus, it is strongly suggested that Tetrahymena converts GTP into unconjugated pteridine derivatives. In this study, dopamine was detected as the major catecholamine, while neither epinephrine nor norepinephrine was identified. Indeed, this protozoa was shown to possess the activity of a dopamine synthesizing enzyme, aromatic L-amino acid decarboxylase. On the other hand, activities of tyrosine hydroxylase or tyrosinase which converts tyrosine into dopa, the substrate of aromatic L-amino acid decarboxylase, could not be detected in this protozoa. Furthermore, neither dopamine beta-hydroxylase activity nor phenylethanolamine N-methyltransferase activity could be identified by the HPLC methods.

Induction of kranz anatomy and C4-like biochemical characteristics in a submerged amphibious plant by abscisic acid

The amphibious leafless sedge Eleocharis vivipara develops C4-like traits as well as Kranz anatomy under terrestrial conditions, but it develops C3-like traits without Kranz anatomy under submerged conditions. When submerged plants are exposed to aerial conditions, they rapidly produce new photosynthetic tissues with C4-like traits. In this study, experiments were performed to determine whether abscisic acid (ABA), a plant stress hormone, could induce the formation of photosynthetic tissues with Kranz anatomy and C4-like biochemical traits under water in the submerged form. When the submerged plants were grown in water containing 5 &mgr;M ABA, they developed new photosynthetic tissues with Kranz anatomy, forming well-developed Kranz (bundle sheath) cells that contained many organelles. The ABA-induced tissues accumulated large amounts of phosphoenolpyruvate carboxylase, pyruvate orthophosphate dikinase, and NAD-malic enzyme at the appropriate cellular sites. The tissues had 3.4 to 3.8 times more C4 enzyme activity than did tissues of the untreated submerged plants. Carbon-14 pulse and carbon-12 chase experiments revealed that the ABA-induced tissues fixed higher amounts of carbon-14 into C4 compounds and lower amounts of carbon-14 into C3 compounds as initial products than did the submerged plants and that they exhibited a C4-like pattern of carbon fixation under aqueous conditions of low carbon, indicating enhanced C4 capacity in the tissues. This report provides an example of the hormonal control of the differentiation of the structural and functional traits required for the C4 pathway.

Dynamic viscoelastic properties in torsion of four commercially available resins for crown and bridge.

In order to clarify the viscoelastic properties of four commercial resins for crown and bridge, the dynamic shear modulus (G'), Knoop hardness (KHN), filler content, quantity of unreacted substance and thermal expansion were measured. The results were as follows. The G' (37 degrees C) of each material ranged from 2.61-11.1 GPa, and gradually decreased with increasing temperature. KHN (23 degrees C) ranged from 29.4 to 120, and the most significant correlation (0.999) was found between G' and KHN. Of the relationship among G', filler content and unreacted substance, there was a highly significant correlation (0.980) between G' and filler content. The coefficient of linear-thermal expansion (alpha) also ranged from 17.9-65.0 x 10(-6)/degree C (25-60 degrees C), and the correlation (-0.961) between G' and alpha was meaningful. It can be presumed that the temperature dependence of G' is closely connected with the specific volume of each material, together with an increase in temperature.

Expression of C3 and C4 photosynthetic characteristics in the amphibious plant Eleocharis vivipara: structure and analysis of the expression of isogenes for pyruvate, orthophosphate dikinase.

Eleocharis vivipara, a unique leafless amphibious sedge, adopts the C4 mode of photosynthesis under terrestrial conditions and the C3 mode under submerged aquatic conditions. To analyze the molecular basis of these responses to the contrasting environments, we isolated and characterized two full-length cDNAs for a key C4 enzyme, pyruvate, orthophosphate dikinase (PPDK; EC 2.7.9.1). The isogenes for PPDK, designated ppdk1 and ppdk2, were highly homologous to one another but not identical. The PPDK1 protein, deduced from the nucleotide sequence of the cDNA, contained an extra domain at the amino terminus which, presumably, serves as a chloroplast transit peptide, while PPDK2 lacked this extra domain. It seems likely, therefore, that the ppdk1 and ppdk2 genes encode a chloroplastic and a cytosolic PPDK, respectively. Genomic Southern blot analysis revealed the existence of a small family of genes for PPDK in the genome of E. vivipara. Northern blot analysis indicate that both chloroplastic and cytosolic genes for PPDK are expressed simultaneously in the culms, a photosynthetic organ, of E. vivipara and that the pattern of expression of these genes differs between the growth forms.

Photosynthesis pathways, ecological characteristics, and the geographical distribution of the Cyperaceae in Japan.

The nature of the photosynthetic pathways of Cyperaceae found in Japan were investigated on the basis of Kranz anatomy, the CO2 compensation concentration and previously reported data. Among 301 species (96% of all cyperaceous species recorded in the region), 58 species were classified as being C4 plants. These C4 species were scattered among the tribes Fimbristylideae, Lipocarpheae, Cypereae and Rhynchosporeae in the subfamily Cyperoideae. The genera Cyperus, Eleocharis and Rhynchospora included, in Japan, both C3 and C4 species within a single genus. Using these data, an analysis was made of the ecological characteristics and geographical distribution of the C3 and C4 species in Japan. Although cyperaceous species grow in markedly different environments, the majority were found in wet and aquatic areas (61%) or shaded areas, such as forest floors (20%). Most of the C3 species were also hygrophytes (58%) and forest-living species (25%), and C3 species growing in mesic and dry areas were relatively rare. The C4 species inhabited wet and aquatic (75%), mesic (13%) and dry areas (6%) and showed marked ecological characteristics with respect to soil-moisture conditions, unlike other C4 plants, although they were absent from shaded habitats. In order to determine the climatic factors that influence the relative floristic abundance of C3 and C4 members of the Cyperaceae in Japan, the ratios of number of C4 species to the total number of members of Cyperaceae (C4 percentage) in 16 representative locales were examined in terms of various climatic variables. There were strong positive correlations between the C4 percentage and temperature. Among the C3 groups of three subfamilies, there were different distributional trends for various temperature regimes. The C3 subfamily Caricoideae increased its relative contribution to the cyperaceous flora with a decrease in mean annual temperature, while the C3 subfamily Sclerioideae exhibited the opposite pattern. The C3 group of the subfamily Cyperoideae did not show any marked change in pattern along temperature gradients, unlike the two other C3 subfamilies, and seemed to be heterogeneous in terms of its response to temperature. The relationships between the C4 biochemical subtypes and ecological characteristics are also discussed.

Effects of altitude acclimatization on pulmonary gas exchange during exercise.

Pulmonary gas exchange was studied in eight normal subjects both before and after 2 wk of altitude acclimatization at 3,800 m (12,470 ft, barometric pressure = 484 Torr). Respiratory and multiple inert gas tensions, ventilation, cardiac output (Q), and hemoglobin concentration were measured at rest and during three levels of constant-load cycle exercise during both normoxia [inspired PO2 (PIO2) = 148 Torr] and normobaric hypoxia (PIO2 = 91 Torr). After acclimatization, the measured alveolar-arterial PO2 difference (A-aPO2) for any given work rate decreased (P less than 0.02). The largest reductions were observed during the highest work rates and were 24.8 +/- 1.4 to 19.7 +/- 0.8 Torr (normoxia) and 22.0 +/- 1.1 to 19.4 +/- 0.7 Torr (hypoxia). This could not be explained by changes in ventilation-perfusion inequality or estimated O2 diffusing capacity, which were unaffected by acclimatization. However, Q for any given work rate was significantly decreased (P less than 0.001) after acclimatization. We suggest that the reduction in A-aPO2 after acclimatization is a result of more nearly complete alveolar/end-capillary diffusion equilibration on the basis of a longer pulmonary capillary transit time.

Pulmonary gas exchange after multiple airway occlusion by beads in the dog.

Patients with asthma frequently show a bimodal distribution of ventilation-perfusion (VA/Q) ratios. One mode is centered on a VA/Q ratio of near normal value, whereas the other has a very low VA/Q ratio. There is usually almost no perfusion of unventilated units. We have suggested that this bimodal pattern is caused by complete obstruction of some airways with ventilation of their distal alveoli through collateral channels. To test this hypothesis we occluded airways of anesthetized dogs with beads of 1.6, 2.4, and 4.8 mm in diameter, and measured VA/Q distributions using the multiple inert gas elimination technique. Beads were inserted via a bronchoscope until the Pao2 fell by 20 mm Hg, or its value was less than 70 mm Hg. Occlusion of small airways (1.6 mm diameter) resulted in a mild increase in dispersion of VA/Q ratios. With 2.4 mm beads, the dispersion increased further. By contrast, all dogs given 4.8 mm beads showed a bimodal distribution of VA/Q ratios. All animals developed some shunt. We interpret these results as showing that when 1.6 mm airways are occluded, collateral ventilation to distal units is so efficient that ventilation is only mildly impaired; however, as occlusion becomes more proximal, collateral ventilation is less effective until with 4.8 mm beads, the ventilation of distal units is so poor that a bimodal pattern develops. The results suggest that the bimodal distributions seen in asthma are compatible with complete obstruction of some airways, and that the levels of obstruction may affect the pattern of VA/Q distribution.

Evidence for tissue diffusion limitation of VO2max in normal humans.

We recently found [at approximately 90% maximal O2 consumption (VO2max)] that as inspiratory PO2 (PIO2) was reduced, VO2 and mixed venous PO2 (PVO2) fell together along a straight line through the origin, suggesting tissue diffusion limitation of VO2max. To extend these observations to VO2max and directly examine effluent venous blood from muscle, six normal men cycled at VO2max while breathing air, 15% O2 and 12% O2 in random order on a single day. From femoral venous, mixed venous, and radial arterial samples, we measured PO2, PCO2, pH, and lactate and computed mean muscle capillary PO2 by Bohr integration between arterial (PaO2) and femoral venous PO2 (PfvO2). VO2 and CO2 production (VCO2) were measured by expired gas analysis, VO2max averaged 61.5 +/- 6.2 (air), 48.6 +/- 4.8 (15% O2), and 38.1 +/- 4.1 (12% O2) ml.kg-1.min-1. Corresponding values were 16.8 +/- 5.6, 14.4 +/- 5.0, and 12.0 +/- 5.0 Torr for PfVO2; 23.6 +/- 3.2, 19.1 +/- 4.2, and 16.2 +/- 3.5 Torr for PVO2; and 38.5 +/- 5.4, 30.3 +/- 4.1, and 24.5 +/- 3.6 Torr for muscle capillary PO2 (PmCO2). Each of the PO2 variables was linearly related to VO2max (r = 0.99 each), with an intercept not different from the origin. Similar results were obtained when the subjects were pushed to a work load 30 W higher to ensure that VO2max had been achieved. By extending our prior observations 1) to maximum VO2 and 2) by direct sampling of femoral venous blood, we conclude that tissue diffusion limitation of VO2max may be present in normal humans. In addition, since PVO2, PfVO2, and PmCO2 all linearly relate to VO2max, we suggest that whichever of these is most readily obtained is acceptable for further evaluation of the hypothesis.

Effect of N-acetylcysteine on gas exchange after methacholine challenge and isoprenaline inhalation in the dog.

N-acetylcysteine (NAC) has antioxidant and possibly mucolytic properties. To determine whether NAC could be of benefit in acute bronchoconstriction induced by methacholine, 12 of 24 anaesthetized dogs (group 1) received NAC i.v. (loading dose 150 mg.kg-1, then 20 mg.kg-1.hr-1). The other 12 (group 2) received diluent. Nebulized methacholine (1%) was then inhaled until arterial oxygen tension (PaO2) fell to a mean of 5.5 kPa, after which isoprenaline 0.5% was inhaled in six dogs of each group to reverse bronchoconstriction. Over the next 3 h we measured total lung resistance, functional residual capacity (FRC), haemodynamic variables, and pulmonary gas exchange for respiratory and inert gases. After methacholine challenge, lung resistance increased and then fell similarly for both groups, but PaO2 was higher in the NAC group (by 0.6-1.9 kPa) throughout the observation period. The ventilation-perfusion distribution measured by inert gas elimination also showed less abnormality in the NAC treated dogs over this time. Mucus was visible during post-mortem in the large airways in about half of the dogs in both groups, with no significant differences between them. These results show that NAC produces a measurable improvement in gas exchange following methacholine challenge (both with and without subsequent isoprenaline therapy) by mechanisms that remain to be determined.

Photosynthetic characteristics of an amphibious plant, Eleocharis vivipara: Expression of C(4) and C(3) modes in contrasting environments.

Eleocharis vivipara Link, a freshwater amphibious leafless plant belonging to the Cyperaceae can grow in both terrestrial and submersed aquatic conditions. Two forms of E. vivipara obtained from these contrasting environments were examined for the characteristics associated with C(4) and C(3) photosynthesis. In the terrestrial form (delta (13)C values = -13.5 to -15.4 per thousand, where per thousand is parts per thousand), the culms, which are photosynthetic organs, possess a Kranz-type anatomy typical of C(4) plants, and well-developed bundle-sheath cells contain numerous large chloroplasts. In the submersed form (delta (13)C value = -25.9 per thousand), the culms possess anatomical features characteristic of submersed aquatic plants, and the reduced bundle-sheath cells contain only a few small chloroplasts. (14)C pulse-(12)C chase experiments showed that the terrestrial form and the submersed form fix carbon by way of the C(4) pathway, with aspartate (40%) and malate (35%) as the main primary products, and by way of the C(3) pathway, with 3-phosphoglyceric acid (53%) and sugar phosphates (14%) as the main primary products, respectively. The terrestrial form showed photosynthetic enzyme activities typical of the NAD-malic enzyme-C(4) subtype, whereas the submersed form showed decreased activities of key C(4) enzymes and an increased ribulose 1,5-bisphosphate carboxylase (EC 4.1.1.39) activity. These data suggest that this species can differentiate into the C(4) mode under terrestrial conditions and into the C(3) mode under submersed conditions.