Cooccurrence of ScDSP Gene Expression, Cell Death, and DNA Fragmentation in a Marine Diatom, Skeletonema costatum

A novel death-specific gene, ScDSP, was obtained from a death stage subtraction cDNA library of the diatom Skeletonema costatum. The full length of ScDSP cDNA was 921 bp in length, containing a 699-bp open reading frame encoding 232 amino acids and two stretches of 66 and 156 bp in the 5′ and 3′ untranslated regions, respectively. Analysis of the peptide structure revealed that ScDSP contained a signal peptide domain, a transmembrane domain, and a pair of EF-hand motifs. When S. costatum grew exponentially at a rate of 1.3 day⁻¹, the ScDSP mRNA level was at 2 μmol·mole 18S rRNA⁻¹. In contrast, when the culture entered the death phase with a growth rate decreasing to 0.5 day⁻¹, ScDSP mRNA increased dramatically to 668 μmol·mole 18S rRNA⁻¹, and a high degree of DNA fragmentation was simultaneously observed. Under the influence of a light-dark cycle, ScDSP expression in both exponential and stationary phases clearly showed a diel rhythm, but the daily mean mRNA level was significantly higher in the stationary phase. Our results suggest that ScDSP may play a role in the molecular mechanism of self-destructive autolysis in phytoplankton under stress.     

Evidence for alpha-Tocopherol Function in the Electron Transport Chain of Chloroplasts

The effect of three different stable radicals-2,2-diphenyl-1-picrylhydrazyl, 1,3,5-triphenyl-verdazyl, and galvinoxyl-was studied in photosystem II of spinach (Spinacia oleracea) chloroplasts. Inhibition by the three was noted on dimethylbenzoquinone reduction in presence of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and on silicomolybdate reduction in presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) in photosystem II and on the H₂O → methylviologen reaction encompassing both photosystems. Inhibition of all photosystem II reactions except silicomolybdate reduction could be partially restored by α-tocopherol or by 9-ethoxy-α-tocopherone but not by other quinones or radical chasers. On this basis, a functional role for α-tocopherol in the electron transport chain of spinach chloroplasts between the DCMU and DBMIB inhibition sites is postulated.     

Kinetic Parameters of Denitrification in a River Continuum

Kinetic parameters for nitrate reduction in intact sediment cores were investigated by using the acetylene blockage method at five sites along the Swale-Ouse river system in northeastern England, including a highly polluted tributary, R. Wiske. The denitrification rate in sediment containing added nitrate exhibited a Michaelis-Menten-type curve. The concentration of nitrate for half-maximal activity (K_map) by denitrifying bacteria increased on passing downstream from 13.1 to 90.4 μM in the main river, but it was highest (640 μM) in the Wiske. The apparent maximal rate (V_maxap) ranged between 35.8 and 324 μmol of N m⁻² h⁻¹ in the Swale-Ouse (increasing upstream to downstream), but it was highest in the Wiske (1,194 μmol N m⁻² h⁻¹). A study of nitrous oxide (N₂O) production at the same time showed that rates ranged from below the detection limit (0.05 μmol of N₂O-N m⁻² h⁻¹) at the headwater site to 27 μmol of N₂O-N m⁻² h⁻¹ at the downstream site. In the Wiske the rate was up to 570 μmol of N₂O-N m⁻² h⁻¹, accounting for up to 80% of total N gas production.     

Thermotolerance of Leaf Discs from Four Isoprene-Emitting Species Is Not Enhanced by Exposure to Exogenous Isoprene

The effects of exogenously supplied isoprene on chlorophyll fluorescence characteristics were examined in leaf discs of four isoprene-emitting plant species, kudzu (Pueraria lobata [Willd.] Ohwi.), velvet bean (Mucuna sp.), quaking aspen (Populus tremuloides Michx.), and pussy willow (Salix discolor Muhl). Isoprene, supplied to the leaves at either 18 μL L⁻¹ in compressed air or 21 μL L⁻¹ in N₂, had no effect on the temperature at which minimal fluorescence exhibited an upward inflection during controlled increases in leaf-disc temperature. During exposure to 1008 μmol photons m⁻² s⁻¹ in an N₂ atmosphere, 21 μL L⁻¹ isoprene had no effect on the thermally induced inflection of steady-state fluorescence. The maximum quantum efficiency of photosystem II photochemistry decreased sharply as leaf-disc temperature was increased; however, this decrease was unaffected by exposure of leaf discs to 21 μL L⁻¹ isoprene. Therefore, there were no discernible effects of isoprene on the occurrence of symptoms of high-temperature damage to thylakoid membranes. Our data do not support the hypothesis that isoprene enhances leaf thermotolerance.     

Transition from Anoxygenic to Oxygenic Photosynthesis in a Microcoleus chthonoplastes Cyanobacterial Mat

Benthic cyanobacterial mats with the filamentous Microcoleus chthonoplastes as the dominant phototroph grow in oxic hypersaline environments such as Solar Lake, Sinai. The cyanobacteria are in situ exposed to chemical variations between 200 μmol of sulfide liter⁻¹ at night and 1 atm pO₂ during the day. During experimental H₂S to O₂ transitions the microbial community was shown to shift from anoxygenic photosynthesis, with H₂S as the electron donor, to oxygenic photosynthesis. Microcoleus filaments could carry out both types of photosynthesis concurrently. Anoxygenic photosynthesis dominated at high sulfide levels, 500 μmol liter⁻¹, while the oxygenic reaction became dominant when the sulfide level was reduced below 100 to 300 μmol liter⁻¹ (25 to 75 μmol of H₂S liter⁻¹). An increasing inhibition of the oxygenic photosynthesis was observed upon transition to oxic conditions from increasing sulfide concentrations. Oxygen built up within the Microcoleus layer of the mat even under 5 mmol of sulfide liter⁻¹ (500 μmol of H₂S liter⁻¹) in the overlying water. The implications of such a localized O₂ production in a highly reducing environment are discussed in relation to the evolution of oxygenic photosynthesis during the Proterozoic era.     

Growth and Nitrogen Uptake Kinetics in Cultured Prorocentrum donghaiense

We compared growth kinetics of Prorocentrum donghaiense cultures on different nitrogen (N) compounds including nitrate (NO₃ ⁻), ammonium (NH₄ ⁺), urea, glutamic acid (glu), dialanine (diala) and cyanate. P. donghaiense exhibited standard Monod-type growth kinetics over a range of N concentraions (0.5–500 μmol N L⁻¹ for NO₃ ⁻ and NH₄ ⁺, 0.5–50 μmol N L⁻¹ for urea, 0.5–100 μmol N L⁻¹ for glu and cyanate, and 0.5–200 μmol N L⁻¹ for diala) for all of the N compounds tested. Cultures grown on glu and urea had the highest maximum growth rates (μ_m, 1.51±0.06 d⁻¹ and 1.50±0.05 d⁻¹, respectively). However, cultures grown on cyanate, NO₃ ⁻, and NH₄ ⁺ had lower half saturation constants (K_μ, 0.28–0.51 μmol N L⁻¹). N uptake kinetics were measured in NO₃ ⁻-deplete and -replete batch cultures of P. donghaiense. In NO₃ ⁻-deplete batch cultures, P. donghaiense exhibited Michaelis-Menten type uptake kinetics for NO₃ ⁻, NH₄ ⁺, urea and algal amino acids; uptake was saturated at or below 50 μmol N L⁻¹. In NO₃ ⁻-replete batch cultures, NH₄ ⁺, urea, and algal amino acid uptake kinetics were similar to those measured in NO₃ ⁻-deplete batch cultures. Together, our results demonstrate that P. donghaiense can grow well on a variety of N sources, and exhibits similar uptake kinetics under both nutrient replete and deplete conditions. This may be an important factor facilitating their growth during bloom initiation and development in N-enriched estuaries where many algae compete for bioavailable N and the nutrient environment changes as a result of algal growth.     

Photoreduction and Photophosphorylation with Tris-Washed Chloroplasts

The artificial electron donor compounds p-phenylenediamine (PD), N, N, N′, N′-tetramethyl-p-phenylenediamine (TMPD), and 2,6-dichlorophenol-indophenol (DCPIP) restored the Hill reaction and photophosphorylation in chloroplasts that had been inhibited by washing with 0.8 m tris (hydroxymethyl) aminomethane (tris) buffer, pH 8.0. The tris-wash treatment inhibited the electron transport chain between water and photosystem II and electron donation occurred between the site of inhibition and photosystem II. Photoreduction of nicotinamide adenine dinucleotide phosphate (NADP) supported by 33 μm PD plus 330 μm ascorbate was largely inhibited by 1 μm 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) while that supported by 33 μm TMPD or DCPIP plus ascorbate was relatively insensitive to DCMU. Experiments with the tris-washed chloroplasts indicated that electron donors preferentially donate electrons to photosystem II but in the presence of DCMU the donors (with the exception of PD at low concentrations) could also supply electrons after the DCMU block. The PD-supported photoreduction of NADP showed the relative inefficiency in far-red light characteristic of chloroplast reactions requiring photosystem II. With phosphorylating systems involving electron donors at low concentrations (33 μm donor plus 330 μm ascorbate) photophosphorylation, which occurred with P/e₂ ratios approaching unity, was completely inhibited by DCMU but with higher concentrations of the donor systems, photophosphorylation was only partially inhibited.     

Chlorophyll Turnover in Skeletonema costatum, a Marine Plankton Diatom

[³H]- and δ-[¹⁴C]Aminolevulinic acids were incorporated into the chlorophylls of Skeletonema costatum, a marine plankton diatom. In the stationary phase of growth, the tetrapyrrole-based pigments reached steady-state labeling after 10 hours. Under conditions of exponential cell division and chlorophyll accumulation, ³H was rapidly lost from the labeled chlorophylls and was replaced with ¹⁴C derived from δ-[4−¹⁴C]aminolevulinic acid. The kinetics of isotope dilution suggests recycling of tetrapyrrole precursors and/or two pigment pools, containing both chlorophyll a and chlorophyllide c, one which turns over rapidly (10 hours) and another which turns over more slowly (100 hours). Calculation of turnover times varied from 3 to 10 hours for chlorophyll a and from 7 to 26 hours for chlorophyllide c. The data suggest the dynamics of chlorophyll metabolism in S. costatum and explain the diatom's ability to undergo light-shade adaptation within a generation time.     

amoA Gene Abundances and Nitrification Potential Rates Suggest that Benthic Ammonia-Oxidizing Bacteria and Not Archaea Dominate N Cycling in the Colne Estuary,United Kingdom

Nitrification, mediated by ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA), is important in global nitrogen cycling. In estuaries where gradients of salinity and ammonia concentrations occur, there may be differential selections for ammonia-oxidizer populations. The aim of this study was to examine the activity, abundance, and diversity of AOA and AOB in surface oxic sediments of a highly nutrified estuary that exhibits gradients of salinity and ammonium. AOB and AOA communities were investigated by measuring ammonia monooxygenase (amoA) gene abundance and nitrification potentials both spatially and temporally. Nitrification potentials differed along the estuary and over time, with the greatest nitrification potentials occurring mid-estuary (8.2 μmol N grams dry weight [gdw]⁻¹ day⁻¹ in June, increasing to 37.4 μmol N gdw⁻¹ day⁻¹ in January). At the estuary head, the nitrification potential was 4.3 μmol N gdw⁻¹ day⁻¹ in June, increasing to 11.7 μmol N gdw⁻¹ day⁻¹ in January. At the estuary head and mouth, nitrification potentials fluctuated throughout the year. AOB amoA gene abundances were significantly greater (by 100-fold) than those of AOA both spatially and temporally. Nitrosomonas spp. were detected along the estuary by denaturing gradient gel electrophoresis (DGGE) band sequence analysis. In conclusion, AOB dominated over AOA in the estuarine sediments, with the ratio of AOB/AOA amoA gene abundance increasing from the upper (freshwater) to lower (marine) regions of the Colne estuary. These findings suggest that in this nutrified estuary, AOB (possibly Nitrosomonas spp.) were of major significance in nitrification.     

Slow degradation of the d1 protein is related to the susceptibility of low-light-grown pumpkin plants to photoinhibition

Photoinhibition of photosystem II (PSII) electron transport and subsequent degradation of the D1 protein were studied in pumpkin (Cucurbita pepo L.) leaves developed under high (1000 μmol m⁻² s⁻¹) and low (80 μmol m⁻² s⁻¹) photon flux densities. The low-light leaves were more susceptible to high light. This difference was greatly diminished when illumination was performed in the presence of chloramphenicol, indicating that a poor capacity to repair photodamaged PSII centers is decisive in the susceptibility of low-light leaves to photoinhibition. In fact, the first phases of the repair cycle, degradation and removal of photodamaged D1 protein from the reaction center complex, occurred slowly in low-light leaves, whereas in high-light leaves the degradation of the D1 protein more readily followed photoinhibition of PSII electron transport. A modified form of the D1 protein, with slightly slower electrophoretic mobility than the original D1, accumulated in the appressed thylakoid membranes of low-light leaves during illumination and was subsequently degraded only slowly.     

Transport, Compartmentation, and Metabolism of Homoserine in Higher Plant Cells : Carbon-13- and Phosphorus-31-Nuclear Magnetic Resonance Studies

The transport, compartmentation, and metabolism of homoserine was characterized in two strains of meristematic higher plant cells, the dicotyledonous sycamore (Acer pseudoplatanus) and the monocotyledonous weed Echinochloa colonum. Homoserine is an intermediate in the synthesis of the aspartate-derived amino acids methionine, threonine (Thr), and isoleucine. Using ¹³C-nuclear magnetic resonance, we showed that homoserine actively entered the cells via a high-affinity proton-symport carrier (K_m approximately 50–60 μm) at the maximum rate of 8 ± 0.5 μmol h⁻¹ g⁻¹ cell wet weight, and in competition with serine or Thr. We could visualize the compartmentation of homoserine, and observed that it accumulated at a concentration 4 to 5 times higher in the cytoplasm than in the large vacuolar compartment. ³¹P-nuclear magnetic resonance permitted us to analyze the phosphorylation of homoserine. When sycamore cells were incubated with 100 μm homoserine, phosphohomoserine steadily accumulated in the cytoplasmic compartment over 24 h at the constant rate of 0.7 μmol h⁻¹ g⁻¹ cell wet weight, indicating that homoserine kinase was not inhibited in vivo by its product, phosphohomoserine. The rate of metabolism of phosphohomoserine was much lower (0.06 μmol h⁻¹ g⁻¹ cell wet weight) and essentially sustained Thr accumulation. Similarly, homoserine was actively incorporated by E. colonum cells. However, in contrast to what was seen in sycamore cells, large accumulations of Thr were observed, whereas the intracellular concentration of homoserine remained low, and phosphohomoserine did not accumulate. These differences with sycamore cells were attributed to the presence of a higher Thr synthase activity in this strain of monocot cells.     

Chlorophyll a Fluorescence and Photosynthetic and Growth Responses of Pinus radiata to Phosphorus Deficiency, Drought Stress, and High CO(2)

Needles from phosphorus deficient seedlings of Pinus radiata D. Don grown for 8 weeks at either 330 or 660 microliters CO₂ per liter displayed chlorophyll a fluorescence induction kinetics characteristic of structural changes within the thylakoid chloroplast membrane, i.e. constant yield fluorescence (F_O) was increased and induced fluorescence ([F_P-F_I]/F_O) was reduced. The effect was greatest in the undroughted plants grown at 660 μl CO₂ L⁻¹. By week 22 at 330 μl CO₂ L⁻¹ acclimation to P deficiency had occurred as shown by the similarity in the fluorescence characteristics and maximum rates of photosynthesis of the needles from the two P treatments. However, acclimation did not occur in the plants grown at 660 μl CO₂ L⁻¹. The light saturated rate of photosynthesis of needles with adequate P was higher at 660 μl CO₂ L⁻¹ than at 330 μl CO₂ L⁻¹, whereas photosynthesis of P deficient plants showed no increase when grown at the higher CO₂ concentration. The average growth increase due to CO₂ enrichment was 14% in P deficient plants and 32% when P was adequate. In drought stressed plants grown at 330 μl CO₂ L⁻¹, there was a reduction in the maximal rate of quenching of fluorescence (R_Q) after the major peak. Constant yield fluorescence was unaffected but induced fluorescence was lower. These results indicate that electron flow subsequent to photosystem II was affected by drought stress. At 660 μl CO₂ L⁻¹ this response was eliminated showing that CO₂ enrichment improved the ability of the seedlings to acclimate to drought stress. The average growth increase with CO₂ enrichment was 37% in drought stressed plants and 19% in unstressed plants.     

Identification of a High-Affinity Phosphate Transporter Gene in a Prasinophyte Alga, Tetraselmis chui, and Its Expression under Nutrient Limitation

A high-affinity phosphate transporter gene, TcPHO, was isolated from a growth-dependent subtracted cDNA library of the marine unicellular alga Tetraselmis chui. The full-length cDNA of TcPHO obtained by 5′ and 3′ rapid amplification of cDNA ends was 1,993 bp long and encoded an open reading frame consisting of 610 amino acids. The deduced amino acid sequence of TcPHO exhibited 51.6 and 49.8% similarity to the amino acid sequences of PHO89 from Saccharomyces cerevisiae and PHO4 from Neurospora crassa, respectively. In addition, hydrophobicity and secondary structure analyses revealed 12 conserved transmembrane domains that were the same as those found in PHO89 and PHO4. The expression of TcPHO mRNA was dependent on phosphate availability. With a low-phosphate treatment, the TcPHO mRNA concentration increased sharply to 2.72 fmol μg of total RNA⁻¹ from day 1 to day 2 and remained at this high level from days 2 to 4. Furthermore, rescue treatment with either phosphate or p-nitrophenyl phosphate effectively inhibited TcPHO mRNA expression. In contrast, TcPHO mRNA expression stayed at a low level (range, 0.25 to 0.28 fmol μg of total RNA⁻¹) under low-nitrate conditions. The expression pattern suggests that TcPHO can be used as a molecular probe for monitoring phosphorus stress in T. chui.     

Fast Fluorescence Quenching from Isolated Guard Cell Chloroplasts of Vicia faba Is Induced by Blue Light and Not by Red Light

Chlorophyll a fluorescence transients from isolated Vicia faba guard cell chloroplasts were used to probe the response of these organelles to light quality. Guard cell chloroplasts were isolated from protoplasts by passing them through a 10-μm nylon net. Intact chloroplasts were purified on a Percoll gradient. Chlorophyll a fluorescence transients induced by actinic red or blue light were measured with a fluorometer equipped with a measuring beam. Actinic red light induced a monophasic quenching, and transients induced by blue light showed biphasic kinetics having a slow and a fast component. The difference between the red and blue light-induced transients could be observed over a range of fluence rates tested (200-800 μmol m⁻² s⁻¹). The threshold fluence rate of blue light for the induction of the fast component of quenching was 200 μmol m⁻² s⁻¹, but in the presence of saturating red light, fluence rates as low as 25 μmol m⁻² s⁻¹ induced the fast quenching. These results indicate that guard cell chloroplasts have a specific response to blue light.     

Successional Development of Sulfate-Reducing Bacterial Populations and Their Activities in a Wastewater Biofilm Growing under Microaerophilic Conditions

A combination of fluorescence in situ hybridization, microprofiles, denaturing gradient gel electrophoresis of PCR-amplified 16S ribosomal DNA fragments, and 16S rRNA gene cloning analysis was applied to investigate successional development of sulfate-reducing bacteria (SRB) community structure and in situ sulfide production activity within a biofilm growing under microaerophilic conditions (dissolved oxygen concentration in the bulk liquid was in the range of 0 to 100 μM) and in the presence of nitrate. Microelectrode measurements showed that oxygen penetrated 200 μm from the surface during all stages of biofilm development. The first sulfide production of 0.32 μmol of H₂S m⁻² s⁻¹ was detected below ca. 500 μm in the 3rd week and then gradually increased to 0.70 μmol H₂S m⁻² s⁻¹ in the 8th week. The most active sulfide production zone moved upward to the oxic-anoxic interface and intensified with time. This result coincided with an increase in SRB populations in the surface layer of the biofilm. The numbers of the probe SRB385- and 660-hybridized SRB populations significantly increased to 7.9 × 10⁹ cells cm⁻³ and 3.6 × 10⁹ cells cm⁻³, respectively, in the surface 400 μm during an 8-week cultivation, while those populations were relatively unchanged in the deeper part of the biofilm, probably due to substrate transport limitation. Based on 16S rRNA gene cloning analysis data, clone sequences that related to Desulfomicrobium hypogeium (99% sequence similarity) and Desulfobulbus elongatus (95% sequence similarity) were most frequently found. Different molecular analyses confirmed that Desulfobulbus, Desulfovibrio, and Desulfomicrobium were found to be the numerically important members of SRB in this wastewater biofilm.     

Biodegradation of Chloromethane by Pseudomonas aeruginosa Strain NB1 under Nitrate-Reducing and Aerobic Conditions

Pseudomonas aeruginosa strain NB1 uses chloromethane (CM) as its sole source of carbon and energy under nitrate-reducing and aerobic conditions. The observed yield of NB1 was 0.20 (±0.06) (mean ± standard deviation) and 0.28 (±0.01) mg of total suspended solids (TSS) mg of CM⁻¹ under anoxic and aerobic conditions, respectively. The stoichiometry of nitrate consumption was 0.75 (±0.10) electron equivalents (eeq) of NO₃⁻ per eeq of CM, which is consistent with the yield when it is expressed on an eeq basis. Nitrate was stoichiometrically converted to dinitrogen (0.51 ± 0.05 mol of N₂ per mol of NO₃⁻). The stoichiometry of oxygen use with CM (0.85 ± 0.21 eeq of O₂ per eeq of CM) was also consistent with the aerobic yield. Stoichiometric release of chloride and minimal accumulation of soluble metabolic products (measured as chemical oxygen demand) following CM consumption, under anoxic and aerobic conditions, indicated complete biodegradation of CM. Acetylene did not inhibit CM use under aerobic conditions, implying that a monooxygenase was not involved in initiating aerobic CM metabolism. Under anoxic conditions, the maximum specific CM utilization rate (k) for NB1 was 5.01 (±0.06) μmol of CM mg of TSS⁻¹ day⁻¹, the maximum specific growth rate (μ_max) was 0.0506 day⁻¹, and the Monod half-saturation coefficient (K_s) was 0.067 (±0.004) μM. Under aerobic conditions, the values for k, μ_max, and K_s were 10.7 (±0.11) μmol of CM mg of TSS⁻¹ day⁻¹, 0.145 day⁻¹, and 0.93 (±0.042) μM, respectively, indicating that NB1 used CM faster under aerobic conditions. Strain NB1 also grew on methanol, ethanol, and acetate under denitrifying and aerobic conditions, but not on methane, formate, or dichloromethane.     

Sorbitol-6-Phosphate Dehydrogenase Expression in Transgenic Tobacco : High Amounts of Sorbitol Lead to Necrotic Lesions

We analyzed transgenic tobacco (Nicotiana tabacum L.) expressing Stpd1, a cDNA encoding sorbitol-6-phosphate dehydrogenase from apple, under the control of a cauliflower mosaic virus 35S promoter. In 125 independent transformants variable amounts of sorbitol ranging from 0.2 to 130 μmol g⁻¹ fresh weight were found. Plants that accumulated up to 2 to 3 μmol g⁻¹ fresh weight sorbitol were phenotypically normal, with successively slower growth as sorbitol amounts increased. Plants accumulating sorbitol at 3 to 5 μmol g⁻¹ fresh weight occasionally showed regions in which chlorophyll was partially lost, but at higher sorbitol amounts young leaves of all plants lost chlorophyll in irregular spots that developed into necrotic lesions. When sorbitol exceeded 15 to 20 μmol g⁻¹ fresh weight, plants were infertile, and at even higher sorbitol concentrations the primary regenerants were incapable of forming roots in culture or soil. In mature plants sorbitol amounts varied with age, leaf position, and growth conditions. The appearance of lesions was correlated with high sorbitol, glucose, fructose, and starch, and low myo-inositol. Supplementing myo-inositol in seedlings and young plants prevented lesion formation. Hyperaccumulation of sorbitol, which interferes with inositol biosynthesis, seems to lead to osmotic imbalance, possibly acting as a signal affecting carbohydrate allocation and transport.     

Purification and partial characterization of a membrane-associated lipoxygenase in tomato fruit

Membrane-associated lipoxygenase from green tomato (Lycopersicon esculentum L. cv Caruso) fruit has been purified 49-fold to a specific activity of 8.3 μmol·min⁻¹·mg⁻¹ of protein by solubilization of microsomal membranes with Triton X-100, followed by anion- exchange and size-exclusion chromatography. The apparent molecular mass of the enzyme was estimated to be 97 and 102 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, respectively. The purified membrane lipoxygenase preparation consisted of a single major band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which cross-reacts with immunoserum raised against soluble soybean lipoxygenase 1. It has a pH optimum of 6.5, an apparent K_m of 6.2 μm, and V_max of 103. μmol·min⁻¹·mg⁻¹ of protein with linoleic acid as substrate. Corresponding values for the partially purified soluble lipoxygenase from tomato are 3.8 μm and 1.3 μmol·min⁻¹·mg⁻¹ of protein, respectively. Thus, the membrane-associated enzyme is kinetically distinguishable from its soluble counterpart. Sucrose density gradient fractionation of the isolated membranes indicated that the membrane-associated lipoxygenase sediments with thylakoids. A lipoxygenase band with a corresponding apparent mol wt of 97,000 was identified immunologically in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins of purified thylakoids prepared from intact chloroplasts isolated from tomato leaves and fruit.     

Carbon gain and photosynthetic response of chrysanthemum to photosynthetic photon flux density cycles

Most models of carbon gain as a function of photosynthetic irradiance assume an instantaneous response to increases and decreases in irradiance. High- and low-light-grown plants differ, however, in the time required to adjust to increases and decreases in irradiance. In this study the response to a series of increases and decreases in irradiance was observed in Chrysanthemum × morifolium Ramat. “Fiesta” and compared with calculated values assuming an instantaneous response. There were significant differences between high- and low-light-grown plants in their photosynthetic response to four sequential photosynthetic photon flux density (PPFD) cycles consisting of 5-minute exposures to 200 and 400 micromoles per square meter per second (μmol m⁻²s⁻¹). The CO₂ assimilation rate of high-light-grown plants at the cycle peak increased throughout the PPFD sequence, but the rate of increase was similar to the increase in CO₂ assimilation rate observed under continuous high-light conditions. Low-light leaves showed more variability in their response to light cycles with no significant increase in CO₂ assimilation rate at the cycle peak during sequential cycles. Carbon gain and deviations from actual values (percentage carbon gain over- or underestimation) based on assumptions of instantaneous response were compared under continuous and cyclic light conditions. The percentage carbon gain overestimation depended on the PPFD step size and growth light level of the leaf. When leaves were exposed to a large PPFD increase, the carbon gain was overestimated by 16 to 26%. The photosynthetic response to 100 μmol m⁻² s⁻¹ PPFD increases and decreases was rapid, and the small overestimation of the predicted carbon gain, observed during photosynthetic induction, was almost entirely negated by the carbon gain underestimation observed after a decrease. If the PPFD cycle was 200 or 400 μmol m⁻² s⁻¹, high- and low-light leaves showed a carbon gain overestimation of 25% that was not negated by the underestimation observed after a light decrease. When leaves were exposed to sequential PPFD cycles (200-400 μmol m⁻² s⁻¹), carbon gain did not differ from leaves exposed to a single PPFD cycle of identical irradiance integral that had the same step size (200-400-200 μmol m⁻² s⁻¹) or mean irradiance (200-300-200 μmol m⁻² s⁻¹).