Dehydration of earthworm cocoons exposed to cold: a novel cold hardiness mechanism

Mechanisms involved in cold hardiness of cocoons of the lumbricid earthworm Dendrobaena octaedra were elucidated by osmometric and calorimetric studies of water relations in cocoons exposed to subzero temperatures. Fully hydrated cocoons contained ca. 3 g water · g dry weight^-1; about 15% of this water (0.5 g·g dry weight^-1) was osmotically inactive or bound. The melting point of the cocoon fluids in fully hydrated cocoons was-0.20°C. Exposure to frozen surroundings initially resulted in supercooling of the cocoons dehydrated (as a result of the vapour pressure difference at a given temperature between supercooled water and ice) to an extent where the vapour pressure of water in the body fluids was in equilibrium with the surrounding ice. This resulted in a profound dehydration of the cocoons, even at mild freezing exposures, and a concomitant slight reduction in the amount of osmotically inactive water. At temperatures around-8°C, which cocoons readily survive, almost all (>97%) osmotically active water had been withdrawn from the cocoons. It is suggested that cold injuries in D. octaedra cocoons observed at still lower temperatures may be related to the degree of dehydration, and possibly to the loss of all osmotically active water. The study indicates that ice formation in the tissues is prevented by equilibrating the body fluid melting point with the exposure temperature. This winter survival mechanism does not conform with the freeze tolerance/freeze avoidance classification generally applied to cold-hardy poikilotherms. Implications of this cold hardiness mechanism for other semi-terrestrial invertebrates are discussed.Abbreviations DSC differential scanning calorimetry - dw dry weight - MP melting point(s) - II water potential - R universal gas constant - T absolute temperature - V specific volume of water     

Carotenoid composition and photon-use efficiency of photosynthesis inGossypium hirsutum L. grown under conditions of slightly suboptimum leaf temperatures and high levels of irradiance

Summary Cotton (Gossypium hirsutum L. var. DP 61) was grown at different temperatures during 12-h light periods, with either 1800–2000 mol photons m^–2 s^–1 (high photon flux density, PFD) or 1000–1100 mol m^–2 s^–1 (medium PFD) incident on the plants. Night temperature was 25°C in all experiments. Growth was less when leaf temperatures were below 30°C during illumination, the effect being greater in plants grown with high PFD (Winter and Königer 1991). Leaf pigment composition and the photon-use efficiency of photosynthesis were analysed to assess whether plants grown with high PFD and suboptimal temperatures experienced a higher degree of high irradiance stress during development than those grown with medium PFD. The chlorophyll content per unit area was 3–4 times less, and the content of total carotenoids about 2 times less, with the proportion of the three xanthophylls zeaxanthin + antheraxanthin + violaxanthin being greater in leaves grown at 20–21°C than in leaves grown at 33–34°C. In leaves from plants grown at 21°C and 1800–2000 mol photons m^–2 s^–1, zeaxanthin accounted for as much as 34% of total carotenoids in the middle of the photoperiod, the highest level recorded in this study. This finding is consistent with a protective role of zeaxanthin under conditions of excess light. At the lower temperatures, the photochemical efficiency of photosystem II, measured as the ratio of variable to maximum fluorescence yield (F _V/F _M) after 12-h dark adaptation, was 0.76 in medium PFD plants and 0.75 in high PFD plants compared with 0.83 and 0.79, respectively, at the higher temperatures. The photon-use efficiency of O₂ evolution () based on absorbed light between 630 and 700nm, decreased with decrease in temperature from 0.102 to 0.07 under conditions of high PFD, but remained above 0.1 at medium PFD. Owing to compensatory reactions in these long-term growth experiments, sustained differences inF _V/F _M and were much less pronounced than the differences in chlorophyll content and dry matter, particularly in plants which had developed at high PFD and low temperature. In fact, in these plants, which exhibited pronounced photobleaching, a largely functional photosynthetic apparatus was still maintained in cells adjacent to the lower leaf surfaces. This was indicated by measurements of photon use efficiencies of photosynthetic O₂ evolution with leaves illuminated first at the upper, and then at the lower surface.Abbreviations F _O yield of dark level fluorescence - F _M maximum yield of fluorescence, induced in a pulse of saturating light - F _V yield of variable fluorescence (=F _M-F _o) - PFD photon flux density - _iw photon use efficiency of O₂ evolution based on white (400–700 nm) incident light - _ir photon use efficiency based on red (630–700 nm) incident light - _aw photon use efficiency based on white absorbed light - _ar photon use efficiency based on red absorbed light     

Ex Vitro Phenotype Stability is Affected by In Vitro Cultivation

Plant phenotype stability during ex vitro growth, one of the main requirements of plant micropropagation, was tested on tobacco. Plants cultivated in vitro in the presence of 3 % sucrose under photon flux density (PFD) of 200 mol m^–2 s^–1 (3 % HL plants) showed the best growth and photosynthetic parameters in the course of 7-day acclimation. However, significant change in phenotype of these plants appeared under a decrease in PFD to 50 mol m^–2 s^–1 during further ex vitro growth (in the period of 7^th – 17^th day). Much higher internodia elongation was found in 3 % HL plants in comparison with plants grown in vitro on sucrose media under PFD of 50 mol m^–2 s^–1 (3 % LL) or without sucrose either under PFD of 50 mol m^–2 s^–1 or 200 mol m^–2 s^–1 (0 % LL, 0 % HL). It can be presumed that 3 % HL plants show permanent demand for high PFD. Neither ABA or chlorophyll contents nor de novo thylakoid membrane synthesis were related to the morphogenic effect of low PFD. Changeable contents of hexoses in leaves of 3 % HL and 3 % LL plants were in no direct correlation to the elongated growth.     

Fusarium solani association with branch and trunk cankers on citrus weakened by cold weather in Florida

Injury or inoculation of apparently healthy citrus trunk bark and wood with Fusarium solani immediately prior to and after the December 1983 freeze resulted in cankers similar to natural branch and trunk cankers which formed on trees following the freeze. Fusarium solani was consistently isolated from active lesions of natural and induced cankered bark. Phytophora spp. were not isolated from cankered tissue. Earliest cankers appeared as a water-soaked area under the bark around the point of inoculation, later F. solani sporodochia formed on the bark surface. Older cankers became dry and cracked but produced no gum. Canker size ranged from 10–90 cm vertical diameter in spring 1984 inoculations but were only several cm diameter following late summer inoculations. Active cankers developed again after inoculation following the January 1985 freeze. Similar cankers were produced on Hamlin sweet orange stems inoculated with F. solani.     

Characteristics of transient photosynthesis in Quercus serrata seedlings grown under lightfleck and constant light regimes

Photosynthetic-induction response and light-fleck utilization were investigated for the current-year seedlings of Quercus serrata, a deciduous tree found in temperate regions of Japan. The tree seedlings were grown under three light regimes: a constant low photosynthetic photon flux density (PFD) regime of 50 mol m^–2 s^–1, a constant high PFD regime of 500 mol m^–2 s^–1, and a lightfleck regime with alternated low (lasting 5 s) and high (lasting 35 s) PFD. The photosynthetic-induction response following a sudden increase of PFD from 50 to 500 mol m^–2 s^–1 exhibited two phases: an initial fast increase complete within 3–5 s, and a second slow increase lasting for 15–20 min. Induction times required to reach 50% and 90% of steady-state assimilation rates were significantly shorter in leaves from the constant low PFD than those from the high PFD regime. During the first 60–100 s, the ratio of observed integrated CO₂ uptake to that predicted by assuming that a steady-state assimilation would be achieved instantaneously after the light increase was significantly higher for leaves from the low PFD regime than from the high PFD regime. Lightfleck utilization was examined for various durations of PFD of 500 mol m^–2 s^–1 on a background PFD of 50 mol m^–2 s^–1. Lightfleck utilization efficiency was significantly higher in low PFD leaves than in the high PFD leaves for 5-s and 10-s lightflecks, but showed no difference among different light regimes for 100-s lightflecks. The contribution of post-illumination CO₂ fixation to total carbon gain decreased markedly with increasing lightfleck durations, but exhibited no significant difference among growth regimes. Photosynthetic performances of induction response and lightfleck utilization in leaves from the lightfleck regime were more similar to those in leaves from the low PFD regime. It may be the total daily PFD rather than PFD dynamics in light regimes that affects the characteristics of transient photosynthesis in Q. serrata seedlings.     

Photoinhibition, 77K chlorophyll fluorescence quenching and phosphorylation of the light-harvesting chlorophyll-protein complex of photosystem II in soybean leaves

When the capacity of leaves for orderly dissipation of excitation energy in photosynthesis is exceeded, one mechanism by which the excess energy appears to be dissipated is through a nonradiative decay process. This process is observed as a reversible quenching of chlorophyll fluorescence emission (77K) from both photosystem II and photosystem I which persists in darkness (Demmig and Björkman 1987, Planta 171, 171–184). Fluorescence quenching was induced in soybean (Glycine max (L.) Merr.) leaves by two methods: 1) changing the composition of the gas surrounding the leaf from normal air to 2% O₂, 0% CO₂ at a low, constant photon flux density (PFD=photon fluence rate), and 2) increasing the PFD in the presence of normal air. In either case the quenching was fully reversible after return to the original condition (low PFD, normal air). The half-time of the relaxation of the quenching was in the order of 30 min. Both treatments resulted in reversible dephosphorylation of the light-harvesting chlorophyll-protein complex of photosystem II (LHC-II). Treatment under photoinhibitory conditions (high PFD plus chloramphenicol) also caused dephosphorylation of LHC-II. Therefore, phosphorylation of LHC-II cannot account for the observed fluorescence quenching. In addition, our results indicate that in vivo a factor other than the redox state of the plastoquinone pool controls LHC-II phosphorylation. This factor may be pH, the pH gradient across the thylakoid membranes.Abbreviations and symbols CAP chloramphenicol - F_o, F_M, F_v instantaneous, maximumr variable fluorescence emission - LHC-II light-haryesting chlorophyll-protein complex of PSII - kDa kilodalton - pH pH gradient across the thylakoid membrane - PFD photon flux density (photon fluence rate) - PQ plastoquinone - PSI, PSII photosystem I, II - Q acceptor of PSII C.I.W.-D.P.B. Publication No. 926     

Light and the maintenance of photosynthetic competence in leaves of Populus balsamifera L. during short-term exposures to high concentrations of sulfur dioxide

Leaves of Populus balsamifera grown under full natural sunlight were treated with 0, 1, or 2 l SO₂·1^-1 air under one of four different photon flux densities (PFD). When the SO₂ exposures took place in darkness or at 300 mol photons·m^-2·s^-1, sulfate accumulated to the levels predicted by measurements of stomatal conductance during SO₂ exposure. Under conditions of higher PFD (750 and 1550 mol·m^-2·s^-1), however, the predicted levels of accumulated sulfate were substantially higher than those obtained from anion chromatography of the leaf extracts. Light-and CO₂-saturated capacity as well as the photon yield of photosynthetic O₂ evolution were reduced with increasing concentration of SO₂. At 2 l SO₂·1^-1 air, the greatest reductions in both photosynthetic, capacity and photon yield occurred when the leaves were exposed to SO₂ in the dark, and increasingly smaller reductions in each occurred with increasing PFD during SO₂ exposure. This indicates that the inhibition of photosynthesis resulting from SO₂ exposure was reduced when the exposure occurred under conditions of higher light. The ratio F _v/F _M (variable/maximum fluorescence emission) for photosyntem II (PSII), a measure of the photochemical efficiency of PSII, remained unaffected by exposure of leaves to SO₂ in the dark and exhibited only moderate reductions with increasing PFD during the exposure, indicating that PSII was not a primary site of damage by SO₂. Pretreatment of leaves with SO₂ in the dark, however, increased the susceptibility of PSII to photoinhibition, as such pretreated leaves exhibited much greater reductions inF _V/F _M when transferred to moderate or high light in air than comparable control leaves.Abbreviations and symbols A₁₂₀₀ photosynthetic capacity (CO₂-saturated rate of O₂ evolution at 1200 mol photons·m^-2·s^-1) - F_o instantaneous fluorescence emission - F_M maximum fluorescence emission - F_V variable fluorescence emission - PFD photon flux density (400–700 nm) - PSII photosystem II     

Transfer cells and solute uptake in minor veins of Pisum sativum leaves

Morphometric and physiological studies were conducted to determine whether the wall ingrowths of transfer cells in the minor-vein phloem of Pisum sativum L. leaves increase the capacity of the cells for solute influx. Size and number of wall ingrowths are positively correlated to the photon flux density (PFD) at which the plants are grown. An analysis of plasmodesmatal frequencies indicated that numerous plasmodesmata are present at all interfaces except those between the sieveelement-transfer-cell complex (SE-TCC) and surrounding cells where plasmodesmata are present but few in number. Flux of exogenous sucrose into the SE-TCC was estimated from kinetic profiles of net sucrose influx into leaf discs, quantitative autoradiography, and measurements of sucrose translocation. Flux based both on the saturable (carrier-mediated) and the linear components of influx was 47% greater in leaves of plants grown at high PFD (1000 mol·m^–2·s^–1) than those grown in low PFD (200 mol·m^–2·s^–1) and was paralleled by a 47% increase in SE-TCC plasmalemma surface area. Flux of endogenous photosynthate across the SE-TCC plasmalemma was calculated from carbon balance and morphometric data. The increase in flux in high-light leaves over that in low-light leaves can be explained on the basis of an increase in plasmalemma surface area. In intact leaves, a standing osmotic gradient may facilitate transport of solute into transfer cells with extensive wall elaborations.Abbreviations LPI leaf plastochron index - PCMBS p-chloromercuribenzenesulfonic acid - PFD(s) photon flux density (densities) - SE-TCC sieve-element-transfer-cell complex This research was supported by National Science Foundation Grant DCB-9104159, U.S. Department of Agriculture Competitive Grant 90000854, and Hatch funds.     

Cold hardiness of larvae of the southwestern corn borer, Diatraea grandiosella

The cold-hardening capacity of field-collected larvae from southeast Missouri and laboratory-reared larvae of the southwestern corn borer, Diatraea grandiosella Dyar, was examined. Supercooling points of non-diapause and diapause larvae collected from maize plants grown in Missouri (36°30 N lat.) were ca.-7.0°C. The hemolymph melting points of diapause field larvae (-0.8°C) were significantly lower than those of non-diapause larvae collected in July (-0.5°C). The supercooling points of hemolymph from non-diapause and diapause field larvae ranged randomly from-10° to-18°C. Supercooling points of non-diapause laboratory larvae increased from-13° to-10°C prior to pupation, whereas those of diapause larvae increased similarly before the onset of diapause, but then decreased during diapause to ca.-17°C. No change in supercooling points or capacity to survive in the presence of ice was observed in diapause laboratory larvae acclimated at 4°C for 63 days. Laboratory and field larvae began to freeze at ca.-1.5°C in the presence of ice, but survived to several degrees below their melting points. The high supercooling points of field larvae appeared to be due to the presence of an environmental ice-nucleator. Although data for laboratory larvae indicate sufficiently low supercooling points to permit winter survival in southeastern Missouri, considerable larval mortality occurs in the field due to inoculative freezing and the presence of an ice-nucleator.     

Effects of growth-light quantity,growth-light quality and CO2 concentration on Rubisco deactivation during low PFD or darkness

The effects of CO₂ concentration and the effects of growth-light conditions on Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) deactivation were examined for Spinacea oleracea (spinach). Rubisco deactivation kinetics and the degree that Rubisco activation limited the rise in photosynthesis following an increase in photon flux density (PFD) were determined from gas-exchange time courses. There were no significant differences in the apparent relaxation time for Rubisco deactivation among leaves exposed to high or low CO₂ (50 or 1000 mol mol^-1) and low PFD (170 mol m^-2 s^-1) or darkness. However, when PFD was increased to 1700 mol m^-2 s^-1 following a period of low PFD or darkness, leaves exposed to low CO₂ × low PFD showed a lower contribution to the photosynthetic induction process by the activation of Rubisco than leaves exposed to the other treatments. For the growth-light experiments, spinach was grown under high PFD × high red:far-red ratio (R:FR), low PFD × high R:FR, or low PFD × low R:FR light environments. Leaves that matured under the low PFD × low R:FR treatment showed a lower percent change in photosynthesis due to Rubisco activation than leaves exposed to the other growth-light treatments. However, there were no significant differences among the growth-light treatments in the maximum contribution of Rubisco activation to the induction response or in the apparent relaxation time for Rubisco deactivation during shade events.     

Concurrent measurements of oxygen- and carbon-dioxide exchange during lightflecks inAlocasia macrorrhiza (L.) G. Don

Oxygen and CO₂ exchange were measured concurrently in leaves of shade-grownAlocasia macrorrhiza (L.) G. Don during lightflecks consisting of short periods of high photon flux density (PFD) superimposed on a low-PFD background illumination. Oxygen exchange was measured with a zirconium-oxide ceramic cell in an atmosphere containing 1 600 bar O₂ and 350 bar CO₂. Following an increase in PFD from 10 to 500 mol photons·m^-2·s^-1, O₂ evolution immediately increased to a maximum rate that was about twice as high as the highest CO₂-exchange rates that were observed. Oxygen evolution then decreased over the next 5–10 s to rates equal to the much more slowly increasing rates of CO₂ uptake. When the PFD was decreased at the end of a lightfleck, O₂ evolution decreased nearly instantaneously to the low-PFD rate while CO₂ fixation continued at an elevated rate for about 20 s. When PFD during the lightfleck was at a level that was limiting for steady-state CO₂ exchange, then the O₂-evolution rate was constant during the lightfleck. This observed pattern of O₂ evolution during lightflecks indicated that the maximum rate of electron transport exceeded the maximum rate of CO₂ fixation in these leaves. In noninduced leaves, rates of O₂ evolution for the first fraction of a second were about as high as rates in fully induced leaves, indicating that O₂ evolution and the electron-transport chain are not directly affected by the leaf's induction state. Severalfold differences between induced and noninduced leaves in O₂ evolution during a lightfleck were seen for lightflecks longer than a few seconds where the rate of O₂ evolution appeared to be limited by the utilization of reducing power in CO₂ fixation.Abbreviation PFD photon flux density (of photosynthetically active radiation)     

Light-path length and population density in photoacclimation of Nannochloropsis sp. (Eustigmatophyceae)

Photoacclimation in the marine eustigmatophyte Nannochlropsis sp., used extensively as a food chaincomponent in aquaculture, was studied both in thelaboratory and outdoors. Cell-chlorophyll andcarotenoids were used as markers to assessphotoacclimation to strong light, as well as todecreasing growth irradiance due to cellproliferation. Focusing on practical aspects involvedin mass cultivation, three different approaches wereused as follows: (a) cultures initially exposed to lowlight (150 mol photon m^-2 s^-1) thentransferred to strong light (1000 to 3000 molphoton m^-2 s^-1); (b) initially low celldensity cultures grown in reactors of differentlight-paths, exposed to strong PFD, in the laboratoryand outdoors; (c) initially low or high cell densitycultures exposed to strong light. As has already beenestablished in many reports, cell-chlorophyllrepresented a sensitive parameter in assessing cellresponse to changes in the intensity of the lightsource as well as to modifications in the light regimeto which the cells were exposed. Cell-chlorophyllconcentration sharply decreased initially upontransferring the culture from low PFD cell^-1 tohigh PFD cell^-1 due to either culture dilution(i.e. decrease in cell density and mutual shading) orto an increase in PFD. After some 7 days ofphotoacclimating to 2000 and 3000 mol photonm^-2 s^-1, chlorophyll a content began to riseat a much faster rate than cell number, which alsoincreased in response to the higher irradiance.Cell-chlorophyll in the culture exposed to 2000mol photon m^-2 s^-1 increased afteracclimation earlier and at a faster rate than in theculture exposed to 3000 mol photon m^-2s^-1, indicating the later irradiance affected astronger stress. The length of the reactor's lightpath exerted a decisive effect on cell response tostrong light through its influence on the light regimein the culture. Upon a sharp increase in PFD,carotenoids in the 1-cm reactor increased in muchhigher rate than chlorophyll, compared with the 3-cmlight path reactors. This marked difference in cellresponse to a shift-up in light was attributed to thevast variations in the light regime associated withdifferences in the length of the light path and areal density. Growth oflow cell density cultures ceased temporarily upontransfer to strong light, in contrast with high celldensity cultures transferred to strong light, whichcontinued growth without a lag.     

Superoxide production by thylakoids during chilling and its implication in the susceptibility of plants to chilling-induced photoinhibition

Factors influencing the rate of superoxide (O ₂ ^- ) production by thylakoids were investigated to determine if increased production of the radical was related to injury induced by chilling at a moderate photon flux density (PFD). Plants used were Spinacia oleracea L., Cucumis sativus L. and Nerium oleander L. grown at either 200° C or 45° C. Superoxide production was determined by electron-spin-resonance spectroscopy of the (O ₂ ^- )-dependent rate of oxidation of 2-ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine (OXANOH) to the corresponding oxazolidinoxyl radical, OXANO ·. For all plants, the steady-state rate of O ₂ ^- production by thylakoids, incubated at 25° C and 350 mol photon · m^–2 · s^–1 (moderate PFD) with added ferredoxin and NADP, was between 7.5 and 12.5 mol · (mg chlorophyll)^–1 · h^–1. Incubation at 5° C and a moderate PFD, decreased the rate of O ₂ ^- production 40% and 15% by thylakoids from S. oleracea and 20° C-grown N. oleander, chillinginsensitive plants, but increased the rate by 56% and 5% by thylakoids from C. sativus and 45° C-grown N. oleander, chilling-sensitive plants. For all plants, the addition of either ferredoxin or methyl viologen increased the rate of O ₂ ^- -production at 25° C by 75–100%. With these electron acceptors, lowering the temperature to 5° C caused only a slight decrease in O ₂ ^- production. In the absence of added electron acceptors, thylakoids produced O ₂ ^- at a rate which was about 45% greater than that when ferredoxin and NADP were present. The addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea reduced O ₂ ^- production under all conditions tested. The results show that the rate of O ₂ ^- production increases in thylakoids when the rate of electron transfer to NADP is reduced. This could explain differences in the susceptibility of thylakoids from chilling-sensitive and chilling-insensitive plants to chilling at a moderate PFD, and is consistent with the proposal that O ₂ ^- production is involved in the injury leading to the inhibition of photosynthesis induced under these conditions.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophen-yl)-1,1-dimethylurea - Fd ferredoxin - MV methyl viologen - 20°oleander Nerium oleander grown at 20° C - 45°-oleander N. oleander grown at 45° C - OXANOH 2-ethyl-1-hydroxy-2,5,5-tri-methyl-3-oxazolidine - PFD photon flux density (photon fluence rate) - TEMED tetramethyl ethylenediamine We would like to thank R.T. Furbank, R.S.B.S., Australian National University, Canberra, A.C.T., and C.B. Osmond, now of Duke University, Durham, N.C., USA, for the gift of ferredoxin, R.A.J.H. was supported by a Commonwealth Postgraduate Research Award.     

Photoinhibition of photosynthesis in intact bean leaves: role of light and temperature,and requirement for chloroplast-protein synthesis during recovery

Photoinhibition of photosynthesis was induced in intact leaves of Phaseolus vulgaris L. grown at a photon flux density (PFD; photon fluence rate) of 300 mol·m^-2·s^-1, by exposure to a PFD of 1400 mol·m^-2·s^-1. Subsequent recovery from photoinhibition was followed at temperatures ranging from 5 to 35°C and at a PFD of either 20 or 140 mol·m^-2·s^-1 or in complete darkness. Photoinhibition and recovery were monitored mainly by chlorophyll fluorescence emission at 77K but also by photosynthetic O₂ evolution. The effects of the protein-synthesis inhibitors, cycloheximide and chloramphenicol, on photoinhibition and recovery were also determined. The results demonstrate that recovery was temperature-dependent with rates slow below 15°C and optimal at 30°C. Light was required for maximum recovery but the process was light-saturated at a PFD of 20 mol·m^-2·s^-1. Chloramphenicol, but not cycloheximide, inactivated the repair process, indicating that recovery involved the synthesis of one or more chloroplast-encoded proteins. With chloramphenicol, it was shown that photoinhibition and recovery occurred concomitantly. The temperature-dependency of the photoinhibition process was, therefore, in part determined by the effect of temperature on the recovery process. Consequently, photoinhibition is the net difference between the rate of damage and the rate of repair. The susceptibility of chilling-sensitive plant species to photoinhibition at low temperatures is proposed to result from the low rates of recovery in this temperature range.Abbreviations and symbols Da Dalton - Fo, Fm, Fv instantaneous, maximum, variable fluorescence emission - PFD photon flux density - PSII photosystem II - photon yield C.I.W.-D.P.B. Publication No. 871     

Phase Transitions of Frozen Camu-camu (Myrciaria dubia (H.B.K.) McVaugh) Pulp: Effect of Cryostabilizer Addition

Camu-camu is a tropical fruit with very high vitamin C content and commercialized as frozen pulp. Enthalpies of freezing, temperatures of the onset of ice melting, and glass transition temperatures of the maximally freeze-concentrated phase () of camu-camu pulp and of samples containing maltodextrin (DE20) and sucrose were measured by differential scanning calorimetry. Maltodextrin exhibited the largest freeze stabilization potential, increasing from −58.2 °C (natural pulp) to −39.6 °C when 30% (w/w) maltodextrin DE 20 was added. Sucrose showed negligible effect on but enhanced considerably the freezing point depression and less amount of ice was formed.     

Application of Carrier and Plasticizer to Improve the Dissolution and Bioavailability of Poorly Water-Soluble Baicalein by Hot Melt Extrusion

The objective of this study was to develop a suitable formulation for baicalein (a poorly water-soluble drug exhibiting high melting point) to prepare solid dispersions using hot melt extrusion (HME). Proper carriers and plasticizers were selected by calculating the Hansen solubility parameters, evaluating melting processing condition, and measuring the solubility of obtained melts. The characteristic of solid dispersions prepared by HME was evaluated. The dissolution performance of the extrudates was compared to the pure drug and the physical mixtures. Physicochemical properties of the extrudates were characterized by differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), and Fourier transform infrared spectroscopy (FTIR). Relative bioavailability after oral administration in beagle dogs was assessed. As a result, Kollidon VA64 and Eudragit EPO were selected as two carriers; Cremophor RH was used as the plasticizer. The dissolution of all the extrudates was significantly improved. DSC and PXRD results suggested that baicalein in the extrudates was amorphous. FTIR spectroscopy revealed the interaction between drug and polymers. After oral administration, the relative bioavailability of solid dispersions with VA64 and EPO was comparative, about 2.4- and 2.9-fold greater compared to the pure drug, respectively.

Figure

Open in a separate windowᅟKEY WORDS: baicalein, carrier, high melting point, hot melt extrusion, plasticizer, solid dispersion     

Biochemical and photochemical changes in response to triacontanol in rice (Oryza sativa L.)

Triacontanol (TRIA) increased the contents of total chlorophyll (Chl), Chl a and Chl b by 25.1%, 26.1% and 22.4% respectively 4 h after treatment in rice seedlings. The minimal fluorescence (F₀), the maximal fluorescence (Fm) and Fv/Fm were also higher in TRIA-treated plants. In actinic light, other Chl fluorescence parameters were measured at different photon flux densities (PFD) to construct light response curves of the quantum yield of PSII electron transport (_PSII), light response curves of photochemical quenching (qp), and light response curves of non-photochemical quenching (qN), respectively. The _PSII and qp declined with the increasing PFD with a higher level present in TRIA-treated plants. The qN increased with the increasing PFD with a lower level present in TRIA-treated plants. Two-dimensional gel electrophoresis indicated a protein expression difference between TRIA-treated materials and the controls at the total-soluble-protein level. Rubisco was 30% higher in TRIA-treated plants than in controls. The quantity of other proteins was unchanged in response to TRIA. These data provide biochemical and photochemical evidence for the effects of TRIA on photosynthesis.     

Chlorophyll fluorescence in the resurrection plant Selaginella lepidophylla (Hook. & Grev.) Spring during high-light and desiccation stress,and evidence for zeaxanthin-associated photoprotection

The function of photosystem (PS)II during desiccation and exposure to high photon flux density (PFD) was investigated via analysis of chlorophyll fluorescence in the desert resurrection plant Selaginella lepidophylla (Hook. and Grev.) Spring. Exposure of hydrated, physiologically competent stems to 2000 mol · m^–2 · s^–1 PFD caused significant reductions in both intrinsic fluorescence yield (F_O) and photochemical efficiency of PSII (F_V/F_M) but recovery to pre-exposure values was rapid under low PFD. Desiccation under low PFD also affected fluorescence characteristics. Both F_V/F_M and photochemical fluorescence quenching remained high until about 40% relative water content and both then decreased rapidly as plants approached 0% relative water content. In contrast, the maximum fluorescence yield (F_M) decreased and non-photochemical fluorescence quenching increased early during desiccation. In plants dried at high PFD, the decrease in F_V/F_M was accentuated and F_O was reduced, however, fluorescence characteristics returned to near pre-exposure values after 24-h of rehydration and recovery at low PFD. Pretreatment of stems with dithiothreitol, an inhibitor of zeaxanthin synthesis, accelerated the decline in F_V/F_M and significantly increased F_O relative to controls at 925 mol · m^–2 · s^–1 PFD, and the differences persisted over a 3-h low-PFD recovery period. Pretreatment with dithiothreitol also significantly decreased non-photochemical fluorescence quenching, increased the reduction state of Q_A, the primary electron acceptor of PSII, and prevented the synthesis of zeaxanthin relative to controls when stems were exposed to PFDs in excess of 250 mol · m^–2 · s^–1. These results indicate that a zeaxanthin-associated mechanism of photoprotection exists in this desert pteridophyte that may help to prevent photoinhibitory damage in the fully hydrated state and which may play an additional role in protecting PSII as thylakoid membranes undergo water loss.Abbreviations and Symbols DTT dithiothreitol - EPS epoxidation state - F_O yield of instantaneous fluorescence at open PSII centers - F_M maximum yield of fluorescence at closed PSII centers induced by saturating light - F_M F_M determined during actinic illumination - F_V yield of variable fluorescence (F_M-F_O) - F_V/F_M photochemical efficiency of PSII - q_P photochemical fluorescence quenching - q_NP non-photochemical fluorescence quenching of Schreiber et al. (1986) - NPQ non-photochemical fluorescence quenching from the Stern-Volmer equation - PFD photon flux density - RWC relative water content This paper is based on research done while W.G.E. was on leave of absence at Duke University during the fall of 1990. We would like to thank Dan Yakir, John Skillman, Steve Grace, and Suchandra Balachandran and many others at Duke University for their help and input with this research. Dr. Barbara Demmig-Adams provided zeaxanthin for standard-curve purposes.     

Generation of rhythmic and arrhythmic behaviour of Crassulacean acid metabolism in Kalanchoë daigremontiana under continuous light by varying the irradiance or temperature: Measurements in vivo and model simulations

Leaves of Kalanchoë daigremontiana Hamet et Perr. at a photon flux density (PFD) above 220 mol·m^–2s^–1 (400–700 nm) or at leaf temperatures above 27.0 °C showed a rapid loss of rhythmicity, and a more or less pronounced damping-out of the endogenous circadian rhythm of CO₂ exchange under continuous illumination. This rhythm was reinitiated after reduction of the PFD by 90–120 mol·m^–2·s^–1 or reduction of leaf temperature by 3.5–11.0 °C under otherwise unchanged external conditions. The reduction in the magnitude of the external control parameter of the Crassulacean acid metabolism (CAM) rhythm (i.e. PFD or leaf temperature) set the phase of the new rhythm. The maxima of CO₂ uptake occurred about 5, 28, 51, 75 h after the reduction. Simulations with a CAM model under comparable conditions showed a similar behaviour. The influence of temperature on the endogenous CAM rhythm observed in K. daigremontiana in vivo could be simulated by incorporating into the model temperature-dependent switch modes for passive efflux of malate from the vacuole to the cytoplasm. Thus, the model indicates that tonoplast function plays an important role in regulation of the endogenous CAM rhythm in K. daigremontiana.Abbreviations CAM Crassulacean acid metabolism - PAR photosynthetically active radiation - PFD photon flux density This work was supported by a grant to F.B. and U.L. from Teilprojekt B5 in the Sonderforschungsbereich 199 of the Deutsche Forschungsgemeinschaft (Bonn, Germany) and by a grant to T. E. E. G. from the Sudienstiftung des deutschen Volkes (Bonn, Germany). Erika Ball is thanked for processing of time-course data for the analysis of Fourier spectra.     

Metabolism of 2-carboxyarabinitol 1-phosphate and regulation of ribulose-1,5-bisphosphate carboxylase activity

Metabolism of 2-carboxy-D-arabinitol 1-phosphate (CA1P) is an important component in the light-dependent regulation of ribulose-1,5-bisphosphate carboxylase (Rubisco) activity and whole leaf photosynthetic CO₂ assimilation in many species, and functions as one mechanism for regulating Rubisco activity when photosynthesis is light-limited. Species differ in their capacity to accumulate CA1P, ranging from those which can synthesize levels of this compound approaching or in excess of the Rubisco catalytic site concentration, to those which apparently lack the capacity for CA1P synthesis. CA1P is structurally related to the six carbon transition state intermediate of the carboxylation reaction and binds tightly to the carbamylated catalytic site of Rubisco, making that site unavailable for catalysis. Under steady-state, the concentration of CA1P in the leaf is highest at low photon flux density (PFD) or in the dark. Degradation of CA1P and recovery of Rubisco activity requires light and is stimulated by increasing PFD. The initial degradation reaction is catalyzed by an enzyme located in the chloroplast stroma, CA1P phosphatase, which yields carboxyarabinitol (CA) and inorganic phosphate as its products. The pathway of CA metabolism in the plant remains to be determined. Synthesis of CA1P occurs in the dark, and in Phaseolus vulgaris this process has been shown to be stimulated by low PFD. The pathway of CA1P synthesis and its relationship to the degradative pathway remains unknown at the present time. The discovery of the existence of this previously unknown carbon pathway in photosynthesis indicates that we still have much to learn concerning the regulation of Rubisco activity and photosynthesis.Abbreviations CA 2-carboxy-D-arabinitol - CA1P 2-carboxy-D-arabinitol 1-phosphate - CABP 2-carboxy-D-arabinitol-1,5-bisphosphate (transition state analog) - PFD photon flux density - P₁ inorganic phosphate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) - RuBP ribulose-1,5-bisphosphate