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The alternative oxidase (AOX) pathway of plant mitochondria uncouples respiration from mitochondrial ATP production and may ameliorate plant performance under stressful environmental conditions, such as cold temperatures, by preventing excess accumulation of reactive oxygen species. We tested this model in whole tissues by growing AtAOX1a-transformed Arabidopsis (Arabidopsis thaliana) plants at 12 degrees C. For the first time, to our knowledge, in plants genetically engineered for AOX, we identified a vegetative shoot growth phenotype. Compared with wild type at day 21 after sowing, anti-sense and overexpressing lines showed, on average, 27% reduced leaf area and 25% smaller rosettes versus 30% increased leaf area and 33% larger rosette size, respectively. Lines overexpressing a mutated, constitutively active AOX1a showed smaller phenotypic effects. These phenotypic differences were not the result of a major alteration of the tissue redox state because the changes in levels of lipid peroxidation products, reflecting oxidative damage, and the expression of genes encoding antioxidant and electron transfer chain redox enzymes did not correspond with the shoot phenotypes. However, the observed phenotypes were correlated with the amount of total shoot anthocyanin at low temperature and with the transcription of the flavonoid pathway genes PAL1 and CHS. These results demonstrate that (1) AOX activity plays a role in shoot acclimation to low temperature in Arabidopsis, and that (2) AOX not only functions to prevent excess reactive oxygen species formation in whole tissues under stressful environmental conditions but also affects metabolism through more pervasive effects, including some that are extramitochondrial.

The alternative oxidase (AOX) of plant mitochondria transfers electrons from the ubiquinone pool to oxygen without energy conservation. AOX can use reductant in excess of cytochrome pathway capacity, preventing reactive oxygen species (ROS) formation from an over-reduced ubiquinone pool, and thus may be involved in acclimation to oxidative stresses. The AOX connection with mitochondrial ROS has been investigated only in isolated mitochondria and suspension culture cells. To study ROS and AOX in whole plants, transformed lines of Arabidopsis (Arabidopsis thaliana) were generated: AtAOX1a overexpressors, AtAOX1a anti-sense plants, and overexpressors of a mutated, constitutively active AtAOX1a. In the presence of KCN, leaf tissue of either mutant or wild-type AOX overexpressors showed no increase in oxidative damage, whereas anti-sense lines had levels of damage greater than those observed for untransformed leaves. Similarly, ROS production increased markedly in anti-sense and untransformed, but not overexpressor, roots with KCN treatment. Thus, AOX functions in leaves and roots, as in suspension cells, to ameliorate ROS production when the cytochrome pathway is chemically inhibited. However, in contrast with suspension culture cells, no changes in leaf transcript levels of selected electron transport components or oxidative stress-related enzymes were detected under nonlimiting growth conditions, regardless of transformation type. Further, a microarray study using an anti-sense line showed AOX influences outside mitochondria, particularly in chloroplasts and on several carbon metabolism pathways. These results illustrate the value of expanding AOX transformant studies to whole tissues.

The activity of the alternative pathway is affected by a number of factors, including the level and reduction state of the alternative oxidase (AOX) protein, and the reduction state of the ubiquinone pool. To investigate the significance of these factors for the rate of alternative respiration in vivo, we studied root respiration of six wild monocotyledonous grass species that were grown under identical controlled conditions. The activity of the alternative pathway was determined using the oxygen isotope fractionation technique. In all species, the AOX protein was invariably in its reduced (high activity) state. There was no correlation between AOX activity and AOX protein concentration, ubiquinone (total, reduced, or oxidized) concentration, or the reduction state of the ubiquinone pool. However, when some of these factors are combined in a linear regression model, a good fit to AOX activity is obtained. The function of the AOX is still not fully understood. It is interesting that we found a positive correlation between the activity of the alternative pathway and relative growth rate; a possible explanation for this correlation is discussed. Inhibition of the AOX (with salicylhydroxamic acid) decreases respiration rates less than the activity present before inhibition (i.e. measured with the 18O-fractionation technique).

A postulated role of the CN-resistant alternative respiratory pathway in plants is the maintenance of mitochondrial electron transport at low temperatures that would otherwise inhibit the main phosphorylating pathway and prevent the formation of toxic reactive oxygen species. This role is supported by the observation that alternative oxidase protein levels often increase when plants are subjected to growth at low temperatures. We used oxygen isotope fractionation to measure the distribution of electrons between the main and alternative pathways in mung bean (Vigna radiata) and soybean (Glycine max) following growth at low temperature. The amount of alternative oxidase protein in mung bean grown at 19 degrees C increased over 2-fold in both hypocotyls and leaves compared with plants grown at 28 degrees C but was unchanged in soybean cotyledons grown at 14 degrees C compared with plants grown at 28 degrees C. When the short-term response of tissue respiration was measured over the temperature range of 35 degrees C to 9 degrees C, decreases in the activities of both main and alternative pathway respiration were observed regardless of the growth temperature, and the relative partitioning of electrons to the alternative pathway generally decreased as the temperature was lowered. However, cold-grown mung bean plants that up-regulated the level of alternative oxidase protein maintained a greater electron partitioning to the alternative oxidase when measured at temperatures below 19 degrees C supporting a role for the alternative pathway in response to low temperatures in mung bean. This response was not observed in soybean cotyledons, in which high levels of alternative pathway activity were seen at both high and low temperatures.

Salicylic acid (SA) is a signal in systemic acquired resistance and an inducer of the alternative oxidase protein in tobacco (Nicotiana tabacum cv Xanthi nc) cell suspensions and during thermogenesis in aroid spadices. The effects of SA on the levels of alternative oxidase protein and the pathogenesis-related 1a mRNA (a marker for systemic acquired resistance), and on the partitioning of electrons between the Cyt and alternative pathways were investigated in tobacco. Leaves were treated with 1.0 mM SA and mitochondria isolated at times between 1 h and 3 d after treatment. Alternative oxidase protein increased 2.5-fold within 5 h, reached a maximum (9-fold) after 12 h, and remained at twice the level of control plants after 3 d. Measurements of isotope fractionation of 18O by intact leaf tissue gave a value of 23% at all times, identical to that of control plants, indicating a constant 27 to 30% of electron-flow partitioning to the alternative oxidase independent of treatment with SA. Transgenic NahG tobacco plants that express bacterial salicylate hydroxylase and possess very low levels of SA gave a fractionation of 23% and showed control levels of alternative oxidase protein, suggesting that steady-state alternative oxidase accumulates in an SA-independent manner. Infection of plants with tobacco mosaic virus resulted in an increase in alternative oxidase protein in both infected and systemic leaves, but no increase was observed in comparably infected NahG plants. Total respiration rate and partitioning of electrons to the alternative pathway in virus-infected plants was comparable to that in uninfected controls.

The regulation of electron partitioning between the cytochrome (Cyt) and alternative pathways in soybean (Glycine max L. cv Ransom) mitochondria in the absence of added inhibitors has been studied using the oxygen isotope fractionation technique. This regulation can depend on several factors, including the amount of alternative oxidase protein, the redox status of the alternative oxidase regulatory sulfhydryl-disulfide system, the degree of activation by [alpha]-keto acids, and the concentration and redox state of the ubiquinone pool. We studied electron partitioning onto the alternative pathway in mitochondria isolated from etiolated and light-grown cotyledons and roots to ascertain how these factors interact in different tissues. In light-grown cotyledon mitochondria there is some partitioning to the alternative pathway in state 4, which is increased dramatically by either pyruvate or dithiothreitol. In etiolated cotyledon mitochondria, the alternative pathway shows little ability to compete for electrons with the Cyt pathway under any circumstances. In root mitochondria, control of alternative pathway activity is exercised by both the ubiquinone pool and the regulatory sulfhydryl-disulfide system. In addition, oxygen isotope fractionation by the Cyt and alternative pathways in mitochondria were identical to the fractionation for the respective pathways seen in intact tissue, suggesting that residual respiration is not present in the absence of inhibitors.

Doubling the concentration of atmospheric CO2 often inhibits plant respiration, but the mechanistic basis of this effect is unknown. We investigated the direct effects of increasing the concentration of CO2 by 360 [mu]L L-1 above ambient on O2 uptake in isolated mitochondria from soybean (Glycine max L. cv Ransom) cotyledons. Increasing the CO2 concentration inhibited the oxidation of succinate, external NADH, and succinate and external NADH combined. The inhibition was greater when mitochondria were preincubated for 10 min in the presence of the elevated CO2 concentration prior to the measurement of O2 uptake. Elevated CO2 concentration inhibited the salicylhydroxamic acid-resistant cytochrome pathway, but had no direct effect on the cyanide-resistant alternative pathway. We also investigated the direct effects of elevated CO2 concentration on the activities of cytochrome c oxidase and succinate dehydrogenase (SDH) and found that the activity of both enzymes was inhibited. The kinetics of inhibition of cytochrome c oxidase were time-dependent. The level of SDH inhibition depended on the concentration of succinate in the reaction mixture. Direct inhibition of respiration by elevated CO2 in plants and intact tissues may be due at least in part to the inhibition of cytochrome c oxidase and SDH.

Two soybean [Glycine max (L.) Merr.] lipoxygenase cDNA clones were isolated that represent lipoxygenase genes (designated LOX7 and LOX8) that display increased expression in leaves following wounding. LOX7 and LOX8 were found to be differentially expressed in soybean leaves after wounding. Increased transcript levels of both genes were detected in wounded leaves within 8 h after wounding, but only the expression of LOX7 displayed a systemic wound response. Additionally, the elevated expression of LOX7 in wounded leaves was transient. Twenty-four hours postwounding, LOX7 transcripts were no longer detectable in leaves. In contrast, LOX8 transcript levels were elevated in wounded leaves from 8 to 72 h after wounding. In addition, treatment of soybean plants with methyl jasmonate resulted in higher levels of both LOX7 and LOX8 transcripts in leaves. High levels of expression of both genes were also detected in young leaves, flowers, and immature seed pods, and increases in LOX7 and LOX8 transcripts were observed in leaves following the removal of reproductive sink tissues. The expression of LOX7 and LOX8 in unwounded soybean tissues and increased expression following wounding suggest that the lipoxygenases encoded by these genes may participate in general physiological processes that are enhanced following physical damage.

The contribution of the cyanide-resistant, alternative pathway to plant mitochondrial electron transport has been studied using a modified aqueous phase on-line mass spectrometry-gas chromatography system. This technique permits direct measurement of the partitioning of electrons between the cytochrome and alternative pathways in the absence of added inhibitors. We demonstrate that in mitochondria isolated from soybean (Glycine max L. cv Ransom) cotyledons, the alternative pathway contributes significantly to oxygen uptake under state 4 conditions, when succinate is used as a substrate. However, when NADH is the substrate, addition of pyruvate, an allosteric activator of the alternative pathway, is required to achieve the same level of alternative pathway activity. Under state 3 conditions, when the reduction state of the ubiquinone pool is low, the addition of pyruvate allows the alternative pathway to compete with the cytochrome pathway for electrons from the ubiquinone pool when the cytochrome pathway is not saturated. These results provide direct experimental verification of the kinetics consequences of pyruvate addition on the partitioning of electron flow between the two respiratory pathways. This distribution of electrons between the two unsaturated pathways could not be measured using conventional oxygen electrode methods and illustrates a clear advantage of the mass spectrometry technique. These results have significant ramifications for studies of plant respiration using the oxygen electrode, particularly those studies involving intact tissues.

The levels of individual lipoxygenase isozymes in soybean [Glycine max (L.) Merr.] leaves were assessed during leaf development, after mechanical wounding, and in response to reproductive sink removal. Native isoelectric focusing followed by immunoblotting was employed to examine individual lipoxygenase isozymes. In leaves of all ages, two distinct classes of lipoxygenase isozymes were detected. One class of lipoxygenase isozymes had nearly neutral isoelectric points (pls) ranging from pH 6.8 to 7.2. The other class of lipoxygenase isozymes had acidic pls ranging from pH 4.7 to 5.6. During leaf development, all of the neutral lipoxygenase isozymes and most of the acidic isozymes were present in greatest abundance in the youngest leaves examined and declined in amount as leaf age increased. However, four acidic lipoxygenase isozymes (pl = 4.70, 4.80, 4.90, 4.95) were more abundant in intermediateage leaves than in either the youngest or oldest leaves examined. Following mechanical wounding of leaves, these same four acidic isozymes also increased in abundance both locally and systemically in leaves from wounded plants. Unlike the specific effects of wounding on the lipoxygenase isozymes in leaves, reproductive sink removal stimulated a general increase in most of the acidic lipoxygenase isozymes in leaves.

Evidence for a mixed population of covalently and noncovalently associated dimers of the cyanide-resistant alternative oxidase protein in plant mitochondria is presented. High molecular mass (oxidized) species of the alternative oxidase protein, having masses predicted for homodimers, appeared on immunoblots when the sulfhydryl reductant, dithiothreitol (DTT), was omitted from sodium dodecyl sulfate-polyacrylamide gel sample buffer. These oxidized species were observed in mitochondria from soybean (Glycine max [L.] Merr. cv Ransom), Sauromatum guttatum Schott, and mung bean (Vigna radiata [L.] R. Wilcz). Reduced species of the alternative oxidase were also present in the same mitochondrial samples. The reduced and oxidized species in isolated soybean cotyledon mitochondria could be interconverted by incubation with the sulfhydryl reagents DTT and azodicarboxylic acid bis(dimethylamide) (diamide). Treatment with chemical cross-linkers resulted in cross-linking of the reduced species, indicating a noncovalent dimeric association among the reduced alternative oxidase molecules. Alternative pathway activity of soybean mitochondria increased following reduction of the alternative oxidase protein with DTT and decreased following oxidation with diamide, indicating that electron flow through the alternative pathway is sensitive to the sulfhydryl/disulfide redox poise. In mitochondria from S. guttatum floral appendix tissue, the proportion of the reduced species increased as development progressed through thermogenesis.

A partial purification of the cyanide-resistant, alternative oxidase from skunk cabbage (Symplocarpus foetidus L.) spadix mitochondria is described. Skunk cabbage mitochondria were solubilized in N,N-bis-(3-D-glucon-amido-propyl)deoxycholamide and the alternative oxidase was purified using a batch DEAE-cellulose treatment, followed by precipitation with Extracti-Gel and chromatography on Sephadex G-200. Following pooling and concentrating of the most active fractions from the gel filtration column, a 20- to 30-fold purification of the alternative oxidase was obtained, with no evidence of contamination by cytochrome c oxidase (complex IV) or cytochrome c reductase (complex III). Polyacrylamide gel electrophoresis of the partially purified oxidase showed major polypeptides at 36 and 29 kD, both of which react with monoclonal antibodies raised against the Sauromatum guttatum alternative oxidase. The purified oxidase fraction showed no absorbance in the visible spectral region, and addition of sodium borohydride induced no absorbance changes in the ultraviolet region. The purified alternative oxidase catalyzed the four-electron reduction of oxygen to water in the absence of citrate, but catalyzed an apparent two-electron reduction of oxygen to hydrogen peroxide in the presence of 0.7 M citrate.

Discrimination against (18)O during dark respiration in tissues of Kalanchoë daigremontiana, Medicago sativa, and Glycine max was measured using an on-line system that enabled direct measurements of the oxygen fractionation of samples in a gas-phase leaf disk electrode unit. Discrimination factors for cytochrome pathway respiration were 18.6 to 19.8%(o) for all tissues. However, discrimination in cyanide-resistant respiration was significantly higher in green tissues (30.4-31.2%(o)) compared with nongreen tissues (25.3-25.9%(o)). Using these discrimination factors, the partitioning of electron transport to these pathways was calculated from measurements of discrimination in the absence of inhibitors. Changes in flux through the alternative pathway were measured during the light and dark phases of Crassulacean acid metabolism in leaf disks of K. daigremontiana. The flux of electrons through the alternative pathway was higher during deacidification than during the other phases of Crassulacean acid metabolism. The increase in alternative pathway electron flux accounted for all of the increased respiration in the light phase. Despite this increase, simultaneous measurements of malate concentration and respiratory flux confirm that only a small proportion of the total malate decarboxylation occurs in the mitochondria.

The metabolism of [(13)C]malate was studied in the Crassulacean plant Kalanchoë tubiflora following exposure to (13)CO(2) for 2 hour intervals during a 16 hour dark cycle. Nuclear magnetic resonance spectroscopy of [(13)C]malate extracted from labeled tissue revealed that the transient flux of malate to the mitochondria, estimated by the randomization of [4-(13)C]malate to [1- (13)C]malate by fumarase, varied substantially during the dark period. At both 15 and 25 degrees C, the extent of malate label randomization in the mitochondria was greatest during the early and late parts of the dark period and was least during the middle of the night, when the rate of (13)CO(2) uptake was highest. Randomization of labeled malate continued for many hours after malate synthesis had initially occurred. Internally respired (12)CO(2) also served as a source of carbon for malate formation. At 15 degrees C, 15% of the total malate was formed from respired (12)CO(2), while at 25 degrees C, 49% of the accumulated malate was derived from respired (12)CO(2). Some of the malate synthesized from external (13)CO(2) was also respired during the night. The proportion of the total [(13)C]malate respired during the dark period was similar at 15 and 25 degrees C, and respiration of newly formed [(13)C]malate increased as the night period progressed. These data are discussed with regard to the relative fluxes of malate to the mitochondria and the vacuole during dark CO(2) fixation.

Activity of mitochondria isolated from whole seedlings of Echinochloa crus-galli (L.) Beauv. var oryzicola germinated under aerobic and anaerobic conditions for 5 to 7 days was investigated. Mitochondria from both treatments exhibited good respiratory control and ADP/O ratios. Although O(2) uptake was low in anaerobic mitochondria, activity rapidly increased when the seedlings were transferred to air. Mitochondria from both aerobically and anaerobically grown seedlings of E. crus-galli var oryzicola maintained up to 66% of their initial respiration rate in the presence of both cyanide and salicylhydroxamic acid, and the inhibitory effects of cyanide and azide were additive. In addition, antimycin A was not an effective inhibitor of respiration. Reduced-minus-oxidized absorption spectra revealed that cytochromes a, a(3), and b were reduced to a greater extent and cytochrome c was reduced to a lesser extent in anaerobically germinated seedlings relative to that in aerobically germinated seedlings. An absorption maximum in the cytochrome d region of the spectrum was reduced to the same extent under both germination conditions and an absorption maximum at 577 nm was present only in anaerobically germinated seedlings. Anaerobically germinated seedlings contained 70% of the cytochrome c oxidase activity found in air grown seedlings. Upon exposure to air, the developmental pattern of this enzyme in anaerobically germinated seedlings was similar to air controls. Succinate dehydrogenase activity in anaerobic seedlings was only 45% of the activity found in aerobically germinated seeds, but within 1 hour of exposure to air, the activity had increased to control levels. The results suggest that mitochondria isolated from E. crus-galli var oryzicola differ from other plants studied and that the potential for mitochondrial function during anaerobiosis exists.

The binding of radioactively labeled butyl gallate to sucrose gradient-purified mung bean (Vigna radiata L.) mitochondria was studied. Titrations showed the binding of [(14)C]butyl gallate to the mitochondria consisted of both reversible and irreversible components. The reversible component bound with a dissociation constant of approximately 1 micromolar which was comparable to the observed inhibition constant for the inhibition of the alternative pathway by butyl gallate. The reversible binding of labeled butyl gallate was also prevented by addition of excess, unlabeled salicylhydroxamic acid. The concentration of binding sites associated with reversible butyl gallate binding was around 0.5 nanomole per milligram of mitochondrial protein. These results were consistent with the reversible binding site being associated with the butyl gallate site of inhibition of the cyanide-resistant, alternative electron transfer pathway in mung bean mitochondria. In addition to the reversible butyl gallate binding site, a nonspecific, irreversible association of butyl gallate with the mitochondrial membrane was observed. The latter binding did not readily saturate at high butyl gallate concentrations and was not correlated with butyl gallate inhibition of the alternative pathway.

Oxygen uptake during the first hours of imbibition in intact soybean and mung bean seeds showed a marked sensitivity to potassium cyanide but was unaffected by addition of either salicylhydroxamic acid or propyl gallate. However O(2) uptake by finely ground seed particles was very sensitive to the addition of either compound. The results indicated that O(2) uptake in intact, imbibing seeds was associated with a cyanide-sensitive process, most probably mitochondrial mediated respiration, and not the result of the cyanide-insensitive lipoxygenase activity which was readily detectable in ground seed particles.The antioxidant propyl gallate was found to inhibit specifically alternative pathway electron transfer in isolated mung bean mitochondria. Half-maximal inhibition occurred with 2 to 5 micromolar propyl gallate. Kinetic analysis indicated that propyl gallate inhibition of the alternative pathway occurred at, or very near, the site of inhibition of the alternative pathway by salicylhydroxamic acid.A high level of lipoxygenase activity was found to be associated with washed mitochondria isolated from a variety of etiolated plant tissues. Most of this lipoxygenase activity could be eliminated from mung bean mitochondria if the mitochondria were purified on a discontinuous sucrose gradient. This indicated that the mitochondrial-associated activity was probably the result of nonspecific adsorption of lipoxygenase onto the mitochondrial membranes during isolation.

The ability of 19 structural analogs of propyl gallate to inhibit purified soybean seed (Glycine max [L.] Merr. var. Ransom) lipoxygenase-2 (EC 1.13.11.12) was determined. The results indicate that the o-dihydroxy and not the ester function of propyl gallate is essential for inhibition of lipoxygenase. Catechol thus represents the minimum inhibitory structure. Among those compounds possessing an o-dihydroxy function, the K(i)' for inhibition of lipoxygenase is directly related to the lipophilicity of the inhibitor as measured by the octanol-water partition coefficient. The structural features of propyl gallate necessary for inhibition of lipoxygenase were found to differ from those required for inhibition of the plant mitochondrial alternative pathway. This further supports the concept that the alternative oxidase and lipoxygenase are functionally distinct species.

Lipoxygenase protein and activity were examined during establishment, senescence, and rejuvenation of soybean cotyledons. Lipoxygenase protein, as determined on ;Western' immunoblots, and lipoxygenase-1 and -2/3 activities decreased during mobilization of seed reserves 3 to 9 days following planting. Lipoxygenase-1 activity decreased more rapidly than lipoxygenase-2/3 and was not detectable by 11 days after planting. Lipoxygenase protein increased after day 11 while lipoxygenase-2/3 activity continued to decline. During the later stages of cotyledon senescence, both lipoxygenase protein and lipoxygenase-2/3 activity decreased. Upon rejuvenation, lipoxygenase-2/3 activity, but not that of lipoxygenase-1, increased. These results demonstrate that elevated lipoxygenase activity does not represent a universal characteristic of senescent plant tissue.

[(14)C]butyl gallate was used in binding studies to investigate the cyanide-resistant respiratory pathway in mitochondria isolated from a variety of sources displaying varying levels of cyanide resistance. Highly cyanide-resistant mitochondria were isolated from aroid spadices, while moderately cyanide-resistant mitochondria were isolated from either mung bean (Vigna radiata L.) hypocotyls or carbon dioxide/oxygen/ethylene-treated tubers. Totally cyanide-sensitive mitochondria were isolated from untreated tubers and rat liver. With one exception, all the plant mitochondria showed a reversible butyl gallate binding site which saturated at a level of 1.0 to 2.0 nanomoles per milligram protein. The exception, freshly harvested white potato tubers (<1 month from harvest), showed little specific butyl gallate binding, and also showed no appreciable induction of the cyanide-resistant pathway following carbon dioxide/oxygen/ethylene treatment. Only a low level, linear binding, well below that seen with plant mitochondria, was observed with rat liver mitochondria. Taken together, these results suggest a model for the interaction of the alternative pathway with the cytochrome pathway. In this model, the butyl gallate binding site (alternative oxidase) is a constitutive component in those mitochondria that are capable of developing the alternative pathway, and the binding sites associated with a second, inducible component that functions to couple the oxidase to the cytochrome pathway.

Patterns of CO(2) exchange in Spanish moss under various experimental conditions were measured using an infrared gas analysis system. Plants were collected from a study site in North Carolina and placed in a gas exchange chamber for several days of continuous measurements. No substantial seasonal effects on CO(2) exchange were observed. High rates of nocturnal CO(2) uptake were observed under day/night temperature regimes of 25/10, 25/15, 25/20, 30/20, and 35/20 C; however, daytime temperatures of 40 C eliminated nighttime CO(2) uptake and a nighttime temperature of 5 C eliminated nocturnal CO(2) uptake, regardless of day temperature. Constant chamber conditions also inhibited nocturnal CO(2) uptake. Constant high relative humidity (RH) slightly stimulated CO(2) uptake while low nighttime RH reduced nocturnal CO(2) uptake.Reductions in daytime irradiance to approximately 25% full sunlight had no effect on CO(2) exchange. Continuous darkness resulted in continuous CO(2) loss by the plants, but a CO(2) exchange pattern similar to normal day/night conditions was observed under constant illumination. High tissue water content inhibited CO(2) uptake. Wetting of the tissue at any time of day or night resulted in net CO(2) loss. Abrupt increases in temperature or decreases in RH resulted in sharp decreases in net CO(2) uptake.The results indicate that Spanish moss is tolerant of a wide range of temperatures, irradiances, and water contents. They also indicate that high nighttime RH is a prerequisite for high rates of CO(2) uptake.

(13)C nuclear magnetic resonance spectroscopy of intact leaves of Kalanchoë tubiflora was used to observe Crassulacean acid metabolism in vivo. (13)C signals from C-4 of malate were observed after overnight exposure of leaves to (13)CO(2). Illumination of the labeled leaves resulted in a gradual decrease in the malate signals. After a period of darkness in normal air, (13)C signals were detected in all four carbons of malate in the previously labeled leaves. The (13)C nuclear magnetic resonance spectrum of malate in solution was pH dependent, which allowed an estimation of the vacuolar pH from the whole leaf spectrum. The pH was 4.0 following a 14-hour dark period, but rose to greater than 6.0 after 6 hours of illumination.

A procedure was developed for the partial purification of succinate dehydrogenase from mung bean (Vigna radiata L.) hypocotyls and soybean (Glycine max [L] Merr. v. Ransom) cotyledons. The procedure utilized a Triton X-100 extraction followed by ammonium sulfate precipitation. The final fraction was enriched in two polypeptides with approximate molecular weights of 67,000 and 30,000 daltons, exhibited a pH optima of 7.0 to 7.5, contained a b-type cytochrome, and exhibited the characteristic ferredoxin-type and high potential iron-sulfur protein-type electron paramagnetic resonance signals reported for the iron-sulfur centers of mammalian succinate dehydrogenase. Inhibition constants of 1.15 and 24.6 micromolar for oxaloacetate and malonate, respectively, were obtained.