Transcriptional and metabolic insights into the differential physiological responses of arabidopsis to optimal and supraoptimal atmospheric CO2.

BACKGROUND: In tightly closed human habitats such as space stations, locations near volcano vents and closed culture vessels, atmospheric CO(2) concentration may be 10 to 20 times greater than Earth's current ambient levels. It is known that super-elevated (SE) CO(2) (>1,200 µmol mol(-1)) induces physiological responses different from that of moderately elevated CO(2) (up to 1,200 µmol mol(-1)), but little is known about the molecular responses of plants to supra-optimal [CO(2)]. METHODOLOGY/PRINCIPAL FINDINGS: To understand the underlying molecular causes for differential physiological responses, metabolite and transcript profiles were analyzed in aerial tissue of Arabidopsis plants, which were grown under ambient atmospheric CO(2) (400 µmol mol(-1)), elevated CO(2) (1,200 µmol mol(-1)) and SE CO(2) (4,000 µmol mol(-1)), at two developmental stages early and late vegetative stage. Transcript and metabolite profiling revealed very different responses to elevated versus SE [CO(2)]. The transcript profiles of SE CO(2) treated plants were closer to that of the control. Development stage had a clear effect on plant molecular response to elevated and SE [CO(2)]. Photosynthetic acclimation in terms of down-regulation of photosynthetic gene expression was observed in response to elevated [CO(2)], but not that of SE [CO(2)] providing the first molecular evidence that there appears to be a fundamental disparity in the way plants respond to elevated and SE [CO(2)]. Although starch accumulation was induced by both elevated and SE [CO(2)], the increase was less at the late vegetative stage and accompanied by higher soluble sugar content suggesting an increased starch breakdown to meet sink strength resulting from the rapid growth demand. Furthermore, many of the elevated and SE CO(2)-responsive genes found in the present study are also regulated by plant hormone and stress. CONCLUSIONS/SIGNIFICANCE: This study provides new insights into plant acclimation to elevated and SE [CO(2)] during development and how this relates to stress, sugar and hormone signaling.

Feasibility of ultraviolet-light-emitting diodes as an alternative light source for photocatalysis.

The objective of this study was to determine whether ultraviolet-light-emitting diodes (UV-LEDs) could serve as an efficient photon source for heterogeneous photocatalytic oxidation (PCO). An LED module consisting of 12 high-power UV-A (lambda max = 365 nm) LEDs was designed to be interchangeable with a UV-A fluorescent black light blue (BLB) lamp for a bench scale annular reactor packed with silica-titania composite (STC) pellets. Lighting and thermal properties of the module were characterized to assess its uniformity and total irradiance. A forward current (I(F)) of 100 mA delivered an average irradiance of 4.0 mW cm(-2) at a distance of 8 mm, which is equivalent to the maximum output of the BLB, but the irradiance of the LED module was less uniform than that of the BLB. The LED and BLB reactors were tested for the oxidization of ethanol (50 ppm(v)) in a continuous-flow-through mode with 0.94 sec residence time. At the same average irradiance, the UV-A LED reactor resulted in a lower CO2 production rate (19.8 vs. 28.6 nmol L(-1) s(-1)), lower ethanol removal (80% vs. 91%), and lower mineralization efficiency (28% vs. 44%) than the UV-A BLB reactor. Ethanol mineralization was enhanced with the increase of the irradiance at the catalyst surface. This result suggests that reduced ethanol mineralization in the LED reactor relative to the BLB reactor at the same average irradiance could be attributed to the nonuniform irradiance over the photocatalyst, that is, a portion of the catalyst was exposed to less than the average irradiance. The potential of UV-A LEDs may be fully realized by optimizing the light distribution over the catalyst and utilizing their instantaneous "on" and "off" feature for periodic irradiation. Nevertheless, our results also showed that the current UV-A LED module had the same wall plug efficiency (WPE) of 13% as that of the UV-A BLB, demonstrating that UV-A LEDs are a viable photon source both in terms of WPE and PCO efficiency.

Bacterial attraction and quorum sensing inhibition in Caenorhabditis elegans exudates.

Caenorhabditis elegans, a bacterivorous nematode, lives in complex rotting fruit, soil, and compost environments, and chemical interactions are required for mating, monitoring population density, recognition of food, avoidance of pathogenic microbes, and other essential ecological functions. Despite being one of the best-studied model organisms in biology, relatively little is known about the signals that C. elegans uses to interact chemically with its environment or as defense. C. elegans exudates were analyzed by using several analytical methods and found to contain 36 common metabolites that include organic acids, amino acids, and sugars, all in relatively high abundance. Furthermore, the concentrations of amino acids in the exudates were dependent on developmental stage. The C. elegans exudates were tested for bacterial chemotaxis using Pseudomonas putida (KT2440), a plant growth promoting rhizobacterium, Pseudomonas aeruginosa (PAO1), a soil bacterium pathogenic to C. elegans, and Escherichia coli (OP50), a non-motile bacterium tested as a control. The C. elegans exudates attracted the two Pseudomonas species, but had no detectable antibacterial activity against P. aeruginosa. To our surprise, the exudates of young adult and adult life stages of C. elegans exudates inhibited quorum sensing in the reporter system based on the LuxR bacterial quorum sensing (QS) system, which regulates bacterial virulence and other factors in Vibrio fischeri. We were able to fractionate the QS inhibition and bacterial chemotaxis activities, thus demonstrating that these activities are chemically distinct. Our results demonstrate that C. elegans can attract its bacterial food and has the potential of partially regulating the virulence of bacterial pathogens by inhibiting specific QS systems.

Plant secondary metabolism in altered gravity.

Plans by the space program to use plants for food supply and environmental regeneration have led to an examination of how plants grow in microgravity. Because secondary metabolic compounds are so important in determining the nutritional and flavor characteristics of plants-as well as making plants more resistant to biotic and abiotic stresses-their responses to altered gravity are now being studied. These experiments are technically challenging because temperature, humidity, atmospheric composition, light, and water status must be maintained around the plant while simultaneously altering the g-load, either in the free-fall of orbital spacecraft or on a centrifuge rotor. In general, plants have shown increased accumulation of small secondary metabolites in microgravity (<10(-3) g), while these have decreased in hypergravity (>1-g). Gravity-related changes in the plant environment as well as mechanical loading effects account for these responses.

Super-elevated CO2 interferes with stomatal response to ABA and night closure in soybean (Glycine max).

Studies have shown stomatal conductance (g(s)) of plants exposed to super-elevated CO2 (>5000micromol mol(-1)) increases in several species, in contrast to a decrease of g(s) caused by moderate CO2 enrichment. We conducted a series of experiments to determine whether super-elevated CO2 alters stomatal development and/or interferes with stomatal closure in soybean (Glycine max). Plants were grown at nominal ambient (400), elevated (1200) and super-elevated (10,000micromol mol(-1)) CO2 in controlled environmental chambers. Stomatal density of the plant leaf was examined by a scanning electron microscope (SEM), while the stomatal response to the application of exogenous abscisic acid (ABA), a phytohormone associated with water stress and stomatal control, was investigated in intact growing plants by measuring the g(s) of abaxial leaf surfaces using a steady-state porometer. Relative to the control (400micromol mol(-1) CO2) plants, daytime stomatal conductance (g(s,day)) of the plants grown under 1200 and 10,000micromol mol(-1) CO2 was reduced by 38% and 15%, respectively. Dark period stomatal conductance (g(s,night)) was unaffected by growing under 1200mumol mol(-1) CO2) but dramatically increased under 10,000micromol mol(-1) CO2. Stomatal density increased by 10% in the leaves of 10,000micromol mol(-1) CO2-grown plants, which in part contributed to the higher g(s,night) values. Elevating [CO2] to 1200micromol mol(-1) enhanced ABA-induced stomatal closure, but further increasing CO2 to 10,000micromol mol(-1) significantly reduced ABA-induced stomatal closure. These results demonstrated that stomatal response to ABA is CO2 dependent. Hence, a stomatal failure to effectively respond to an ABA signal and to close at night under extremely high CO2 may increase plants susceptibility to other abiotic stresses.

Gravity control of growth form in Brassica rapa and Arabidopsis thaliana (Brassicaceae): Consequences for secondary metabolism.

How gravity influences the growth form and flavor components of plants is of interest to the space program because plants could be used for food and life support during prolonged missions away from the planet, where that constant feature of Earth's environment does not prevail. We used plant growth hardware from prior experiments on the space shuttle to grow Brassica rapa and Arabidopsis thaliana plants during 16-d or 11-d hypergravity treatments on large-diameter centrifuge rotors. Both species showed radical changes in growth form, becoming more prostrate with increasing g-loads (2-g and 4-g). In Brassica, height decreased and stems thickened in a linear relationship with increasing g-load. Glucosinolates, secondary compounds that contribute flavor to Brassica, decreased by 140% over the range of micro to 4-g, while the structural secondary compound, lignin, remained constant at ∼15% (w/w) cell wall dry mass. Stem thickening at 4-g was associated with substantial increases in cell size (47%, 226%, and 33% for pith, cortex, and vascular tissue), rather than any change in cell number. The results, which demonstrate the profound effect of gravity on plant growth form and secondary metabolism, are discussed in the context of similar thigmostresses such as touch and wind.

Influence of Microgravity Environment on Root Growth, Soluble Sugars, and Starch Concentration of Sweetpotato Stem Cuttings.

Because sweetpotato [Ipomoea batatas (L.) Lam.] stem cuttings regenerate very easily and quickly, a study of their early growth and development in microgravity could be useful to an understanding of morphological changes that might occur under such conditions for crops that are propagated vegetatively. An experiment was conducted aboard a U.S. Space Shuttle to investigate the impact of microgravity on root growth, distribution of amyloplasts in the root cells, and on the concentration of soluble sugars and starch in the stems of sweetpotatoes. Twelve stem cuttings of 'Whatley/Loretan' sweetpotato (5 cm long) with three to four nodes were grown in each of two plant growth units filled with a nutrient agarose medium impregnated with a half-strength Hoagland solution. One plant growth unit was flown on Space Shuttle Colombia for 5 days, whereas the other remained on the ground as a control. The cuttings were received within 2 h postflight and, along with ground controls, processed in approximately 45 min. Adventitious roots were counted, measured, and fixed for electron microscopy and stems frozen for starch and sugar assays. Air samples were collected from the headspace of each plant growth unit for postflight determination of carbon dioxide, oxygen, and ethylene levels. All stem cuttings produced adventitious roots and growth was quite vigorous in both ground-based and flight samples and, except for a slight browning of some root tips in the flight samples, all stem cuttings appeared normal. The roots on the flight cuttings tended to grow in random directions. Also, stem cuttings grown in microgravity had more roots and greater total root length than ground-based controls. Amyloplasts in root cap cells of ground-based controls were evenly sedimented toward one end compared with a more random distribution in the flight samples. The concentration of soluble sugars, glucose, fructose, and sucrose and total starch concentration were all substantially greater in the stems of flight samples than those found in the ground-based samples. Carbon dioxide levels were 50% greater and oxygen marginally lower in the flight plants, whereas ethylene levels were similar and averaged less than 10 nL.L (-1). Despite the greater accumulation of carbohydrates in the stems, and greater root growth in the flight cuttings, overall results showed minimal differences in cell development between space flight and ground-based tissues. This suggests that the space flight environment did not adversely impact sweetpotato metabolism and that vegetative cuttings should be an acceptable approach for propagating sweetpotato plants for space applications.

Simultaneous quantification of poly-dispersed anionic, amphoteric and nonionic surfactants in simulated wastewater samples using C18 high-performance liquid chromatography-quadrupole ion-trap mass spectrometry.

This paper describes the development of a guantitative method for direct and simultaneous determination of three frequently encountered surfactants, amphoteric (cocoamphoacetate, CAA), anionic (sodium laureth sulfate, SLES), and nonionic (alcohol ethoxylate, AE) using a reversed-phase C18 HPLC coupled with an ESI ion-trap mass spectrometer (MS). Chemical composition, ionization characteristics and fragmentation pathways of the surfactants are presented. Positive ESI was effective for all three surfactants in agueous methanol buffered with ammonium acetate. The method enables rapid determinations in small sample volumes containing inorganic salts (up to 3.5 g L(-1)) and multiple classes of surfactants with high specificity by applying surfactant specific tandem mass spectrometric strategies. It has dynamic linear ranges of 2-60, 1.5-40, 0.8-56 mg L(-1) with R2 egual or greater than 0.999, 0.98 and 0.999 (10 microL injection) for CAA, SLES, and AE, respectively.

In vitro flavon-3-ol oxidation mediated by a B ring hydroxylation pattern.

Flavonols are potent naturally occurring antioxidants. Chemical oxidation reactions in combination with modern spectroscopic techniques have been employed to identify oxidized flavonoid products. Although many oxidized derivatives have been generated from commercially available starting materials, few studies have developed a sequence of flavonoid substrates with a particular hydroxylation pattern to probe the mechanism of flavonoid oxidation. Here, we use AIBN (2,2'-azobisisobutyronitrile) in combination with a series of hydroxylated flavonols to probe the mechanism of flavonoid dimer formation and the role of singly or doubly oxidized species in generating and promoting oxidized flavonoid products. 3-Methoxyquercetin (3-hydroxyl group blocked) and luteolin (lack of 3-hydroxyl) were reacted with AIBN alone or with a second flavonol to serve as a C-3 hydroxyl donor to examine the mechanism of dimer formation. 3-Hydroxyflavones with increasing hydroxyl substitutions in the B ring were also reacted with AIBN in the presence or absence of an external nucleophile to examine the role of various hydroxyls in the formation of a carbocation intermediate via a doubly oxidized species. The presence of a free C-3 hydroxyl, coupled with a B ring ortho hydroxy unit, appears essential for dimer formation. An increase in the number of hydroxyls in the B ring facilitates products generated from a doubly oxidized species.

Community-level physiological profiling performed with an oxygen-sensitive fluorophore in a microtiter plate.

Community-level physiological profiling based upon fluorometric detection of oxygen consumption was performed on hydroponic rhizosphere and salt marsh litter samples by using substrate levels as low as 50 ppm with incubation times between 5 and 24 h. The rate and extent of response were increased in samples acclimated to specific substrates and were reduced by limiting nitrogen availability in the wells.

Influence of elevated CO(2) on the fungal community in a coastal scrub oak forest soil investigated with terminal-restriction fragment length polymorphism analysis.

Sixteen open-top chambers (diameter, 3.66 m) were established in a scrub oak habitat in central Florida where vegetation was removed in a planned burn prior to chamber installation. Eight control chambers have been continuously exposed to ambient air and eight have been continuously exposed to elevated CO(2) at twice-ambient concentration (approximately 700 ppm) for 5 years. Soil cores were collected from each chamber to examine the influence of elevated atmospheric CO(2) on the fungal community in different soil fractions. Each soil sample was physically fractionated into bulk soil, rhizosphere soil, and roots for separate analyses. Changes in relative fungal biomass were estimated by the ergosterol technique. In the bulk soil and root fractions, a significantly increased level of ergosterol was detected in the elevated CO(2) treatments relative to ambient controls. Fungal community composition was determined by terminal-restriction fragment length polymorphism (T-RFLP) analysis of the internal transcribed spacer (ITS) region. The specificities of different ITS primer sets were evaluated against plant and fungal species isolated from the experimental site. Changes in community composition were assessed by principal component analyses of T-RFLP profiles resolved by capillary electrophoresis. Fungal species richness, defined by the total number of terminal restriction fragments, was not significantly affected by either CO(2) treatment or soil fraction.

Flavonoid oxidation by the radical generator AIBN: a unified mechanism for quercetin radical scavenging.

Four oxidized flavonoid derivatives generated from reacting quercetin (a pentahydroxylated flavone) with the peroxyl radical generator 2,2'-azobis-isobutyronitrile (AIBN) were isolated by chromatographic methods and identified by NMR and MS analyses. Compounds included 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone (2); 1,3,11a-trihydroxy-9-(3,5,7-trihydroxy-4H-1-benzopyran-4-on-2-yl)-5a-(3,4-dihydroxyphenyl)-5,6,11-hexahydro-5,6,11-trioxanaphthacene-12-one (3); 2-(3,4-dihydroxybenzoyloxy)-4,6-dihydroxybenzoic acid (4); and methyl 3,4-dihydroxyphenylglyoxylate (5). Product ratios under different hydrogen ion concentrations and external nucleophiles revealed that two of the products, namely the substituted benzofuranone (2) and the depside (4), are generated from a common carbocation intermediate. Indirect evidence for the operation of a cyclic concerted mechanism in the formation of the dimeric product (3) is provided. The identification of these products supports the model that the principal site of scavenging reactive oxygen species (ROS) in quercetin is the o-dihydroxyl substituent in the B-ring, as well as the C-ring olefinic linkage.

HPLC/ESI-quadrupole ion trap mass spectrometry for characterization and direct quantification of amphoteric and nonionic surfactants in aqueous samples.

An amphoteric (cocamidopropylbetaine, CAPB) and a nonionic (alcohol polyethoxylate, AE) surfactant were characterized by electrospray ionization quadrupole ion trap mass spectrometry (ESI-MS) as to their homologue distribution and ionization/fragmentation chemistry. Quantitative methods involving reversed-phase gradient HPLC and (+)ESI-MSn were developed to directly determine these surfactants in hydroponic plant growth medium that received simulated graywater. The predominant homologues, 12 C alkyl CAPB and 9 EO AE, were monitored to represent the total amount of the respective surfactants. The methods demonstrated dynamic linear ranges of 0.5-250 ng (r2 > 0.996) for CAPB and 8-560 ng (r2 > 0.998) for AE homologue mixture, corresponding to minimum quantification limits of 25 ppb CAPB and 0.4 ppm AE with 20-microL injections. This translated into an even lower limit for individual components due to the polydispersive nature of the surfactants. The procedure was successfully employed for the assessment of CAPB and AE biodegradation in a hydroponic plant growth system used as a graywater bioreactor.