Light-induced changes in the fluorescence yield of chlorophyll a in Anacystis nidulans II. The fast changes and the effect of photosynthetic inhibitors on both the fast and slow fluorescence induction

1. The intensity dependence and spectral variations during thefast transient of chlorophyll a (Chl a) fluorescence have beenanalyzed in the blue-green alga Anacystis nidulans. (Unlikethe case of eukaryotic unicellular green or red algae, the fastfluorescence induction characteristics of the prokaryotic blue-greenalgae had not been documented before.)
2. Dark adapted cellsof Anacystis exhibit two types of fluctuationsin the fluorescenceyield when excited with bright orange light(absorbed mainlyin phycocyanin). The first kinetic patterncalled the fast (sec)fluorescence transient exhibits a characteristicoriginal levelO, intermediary hump I, a pronounced dip D, peakP and a transitorysmall decline to a quasi steady state S.After attaining S,fluorescence yield slowly rises to a maximumlevel M. From M,the decline in fluorescence yield to a terminalT level is extremelyslow as shown earlier by Papageorgiou andGovindjee (8). Ascompared with green and red algae, blue-greenalgae seem tohave a small PS decline and a very characteristicslow SM rise,with a M level much higher than the peak P.
3. A prolonged darkadaptation and relatively high intensity ofexciting illuminationare required to evoke DPS type yield fluctuationsin Anacystis.At low to moderate intensities of exciting light,the time forthe development of P depends on light doses, butfor M, thisremains constant at these intensities.
4. Fluorescence emissionwas heterogeneous during the inductionperiod in Anacystis;the P and the M levels were relativelyenriched in short-wavelengthsystem II Chi a emission as comparedto D and S levels.
5. Thefast DPS transient was found to be affected by electrontransportcofactor (methyl viologen), and inhibitors (e.g.,DCMU, NH₂OH)in a manner suggesting that these changes are mostlyrelatedto the oxido-reduction level of intermediates betweenthe twophotosystems. On the other hand, the slow SM changesin fluorescenceyield, as reported earlier (5, 15), paralleloxygen evolution.These changes were found to be resistant toa variety of electrontransport inhibitors (O-phenanthroline,HOQNO, salicylaldoxime,DCMU, NH₂OH and Antimycin a). It issuggested that, in Anacystis,even in the presence of so-calledinhibitors of cyclic electronflow, a "high energy state" isstill produced.
6. Measurementsof Chlorophyll a fluorescence and delayed lightemission inthe presence of both DCMU and NH₂OH indicate thatthe slow SMchanges are not due to the recovery of the reactioncenter IIin darkness preceeding illumination.
7. Our results, thus, suggestthat in Anacystis a net electrontransport supported oxidation-reductionstate of the quencherQ regulates only partially the developmentof the DPS transient,but the development of the slow fluorescenceyield changes seemsnot to be regulated by these reactions.It appears, from datapresented elsewhere, that the slow risein the yield resultsdue to a structural modification of thethylakoid membrane.

¹We are grateful to the National Science Foundation for financialsupport. (Received November 21, 1972; )     

Gas Exchange and Carbohydrate and Nitrogen Concentrations in Leaves of Pascopyrum smithii (C3) and Bouteloua gracilis (C4) at Different Carbon Dioxide Concentrations and Temperatures

Pascopyrum smithii (C₃) andBouteloua gracilis (C₄) are importantforage grasses native to the Colorado shortgrass steppe. Thisstudy investigated photosynthetic responses of these grassesto long-term CO₂enrichment and temperature in relation to leafnonstructural carbohydrate (TNC) and [N]. Glasshouse-grown seedlingswere transferred to growth chambers and grown for 49 d at twoCO₂concentrations (380 and 750 µmol mol^-1) at 20 and 35°C, and two additional temperatures (25 and 30 °C) at750 µmol mol^-1CO₂. Leaf CO₂exchange rate (CER) was measuredat a plant's respective growth temperature and at two CO₂concentrationsof approx. 380 and 700 µmol mol^-1. Long-term CO₂enrichmentstimulated CER in both species, although the response was greaterin the C₃,P. smithii . Doubling the [CO₂] from 380 to 750 µmolmol^-1stimulated CER ofP. smithii slightly more in plants grownand measured at 30 °C compared to plants grown at 20, 25or 35 °C. CO₂-enriched plants sometimes exhibited lowerCER when compared to ambient-grown controls measured at thesame [CO₂], indicating photosynthetic acclimation to CO₂growthregime. InP. smithii , such reductions in CER were associatedwith increases in TNC and specific leaf mass, reductions inleaf [N] and, in one instance, a reduction in leaf conductancecompared to controls. InB. gracilis , photosynthetic acclimationwas observed more often, but significant changes in leaf metabolitelevels from growth at different [CO₂] were generally less evident.Temperatures considered optimal for growth (C₃: 20 °C; C₄:35 °C) sometimes led to CO₂-induced accumulations of TNCin both species, with starch accumulating in the leaves of bothspecies, and fructans accumulating only inP. smithii. Photosynthesisof both species is likely to be enhanced in future CO₂-enrichedand warmer environments, although responses will sometimes beattenuated by acclimation. Acclimation; blue grama (Bouteloua gracilis (H.B.K.) Lag ex Steud.); leaf nitrogen concentration; nonstructural carbohydrates; photosynthesis; western wheatgrass (Pascopyrum smithii (Rydb.) Love)     

Photophosphorylation in intact algae: Effects of inhibitors, intensity of light, electron acceptor and donors

The luciferin-luciferase method was used to determine ATP extractedfrom darkmaintained and light-exposed samples of the green algaChlorella pyrenoidosa and of the blue-green alga Anacystis nidulans.A few measurements on Synechococcus lividus (a bluegreen thermophile,clone 65?C) are also reported.

1. The light-minus-dark ATP levels (ATP) from aerobic cells ofChlorella and Anacystis were negative; however, ATP from Synechococcuswas positive. Large positive ATP was obtained in regularly grown(RG: moderate light) Chlorella treated with oligomycin; darklevels were reduced, light levels remained essentially unaffected.In high-light exposed (HLE) Chlorella, oligomycin reduced bothlight and dark ATP levels, but positive ATP was still obtained.However, in Anacystis, which has a different organization ofthylakoid membrane, oligomycin severely reduced both the lightand the dark ATP levels and the ATP remained negative.
2. Theoligomycin (12 µM) treated Chlorella and the untreatedAnacystis and Synechococcus show the presence of cyclic photophosphorylationunder conditions in which the non-cyclic electron flow fromphotosystem II to photosystem I is blocked by 10 µM 3-(3,4-dichlorophenyl)-l,l-dimethylurea(DCMU), or not allowed to operate by the absence of CO₂. Cyclicphotophosphorylation ranged from 10–30% of the maximumATP in RG, to 40–50% in HLE Chlorella. In RG Chlorella,cyclic and non-cyclic (in the absence of DCMU) photophosphorylation(ATP) saturate at about 10³ ergs cm^–2 sec^–1 and10⁴ ergs cm^–2 sec^–1 and 10⁴ ergs cm^–2 sec^–1red (>640 nm) light, respectively; a lag was observed inthe light curve.
3. In Chlorella, the addition of the photosystemI electron acceptormethyl viologen (MV; 1 mM) increased ATPby twofold. Furtheraddition of DCMU (25 µm) reduced thisto the level observedwith DCMU alone. If 1 mM reduced dichlorophenolindophenol orphenazine methosulphate (DCPIPH₂ or PMSH₂, respectively)wasadded along with DCMU, the ATP level was 30–40% ofthecontrol. Further addition of MV increased the JATP to be70–80%of that of the control. These and other resultsconfirm thepresence of both non-cyclic and cyclic photophosphorylationin vivo, the former predominating in Chlorella, and the latterin Anacystis and Synechococcus.

(Received May 1, 1973; )     

Growth and Partitioning in Pascopyrum smithii (C3) and Bouteloua gracilis(C4) as Influenced by Carbon Dioxide and Temperature

This study investigated how CO₂and temperature affect dry weight(d.wt) accumulation, total nonstructural carbohydrate (TNC)concentration, and partitioning of C and N among organs of twoimportant grasses of the shortgrass steppe,Pascopyrum smithiiRydb. (C₃) andBouteloua gracilis(H.B.K.) Lag. ex Steud. (C₄).Treatment combinations comprised two temperatures (20 and 35°C)at two concentrations of CO₂(380 and 750 µmol mol^-1),and two additional temperatures of 25 and 30°C at 750 µmolmol^-1CO₂. Plants were maintained under favourable nutrient andsoil moisture and harvested following 21, 35, and 49d of treatment.CO₂-induced growth enhancements were greatest at temperaturesconsidered favourable for growth of these grasses. Comparedto growth at 380 µmol mol^-1CO₂, final d.wt of CO₂-enrichedP.smithiiincreased 84% at 20°C, but only 4% at 35°C. Finald.wt ofB. graciliswas unaffected by CO₂at 20°C, but wasenhanced by 28% at 35°C. Root:shoot ratios remained relativelyconstant across CO₂levels, but increased inP. smithiiwith reductionin temperature. These partitioning results were adequately explainedby the theory of balanced root and shoot activity. Favourablegrowth temperatures led to CO₂-induced accumulations of TNCin leaves of both species, and in stems ofP. smithii, whichgenerally reflected responses of above-ground d.wt partitioningto CO₂. However, CO₂-induced decreases in plant tissue N concentrationswere more evident forP. smithii. Roots of CO₂-enrichedP. smithiihadgreater total N content at 20°C, an allocation of N below-groundthat may be an especially important adaptation for C₃plants.Tissue N contents ofB. graciliswere unaffected by CO₂. Resultssuggest CO₂enrichment may lead to reduced N requirements forgrowth in C₃plants and lower shoot N concentration, especiallyat favourable growth temperatures. Acclimation to CO₂; blue grama; Bouteloua gracilis ; carbohydrate; climate change; global change; grass; growth; growth temperature optima; nitrogen; N uptake; Pascopyrum smithii; western wheatgrass     

Inactivation of the Water-Oxidizing Complex by Exogenous Reductants in PS II Membranes Depleted of Extrinsic Proteins

We have studied the inactivation of the water-oxidizing complexby exogenous, ‘general’ reductants in various typesof PS II membrane. Extraction of the 33, 23 and 17 kDa proteinsfrom PS II membranes rendered the functional Mn susceptibleto rapid solubilization by reductants such as hydroquinone,benzidine and ascorbate, while water analogs, such as NH₂OH,inactivated the complex regardless of the presence of PS IIextrinsic proteins. The extent of the inactivation was dependenton the hydrophobicity of the reductants examined. Diphenylcarbazide,an efficient electron donor to Z⁺ and D⁺, did not inactivatethe Mn complex. As reported earlier [Ghanotakis et al. (1984)Biochim. Biophys. Acta 767: 524], weak illumination deceleratedthe inactivation of the complex by the various reductants. Kineticanalyses of the flash-induced protection provided evidence aboutthe nature of the light state that was not susceptible to thereductants. This state was generated and decayed with half timesof approximately 0.5 and 9 s, respectively. However, such lightprotection was diminished under Cl^–-depleted conditions,at slightly alkaline pH, or when ascorbate was employed as areductant. Furthermore, we observed that the oxidation of N,N,N',N'-tetramethyl-p-phenylenediamine,which reacts with the Mn complex, was accomplished as a biphasicreaction. The amount of the fast phase, which was almost eliminatedafter the reconstitution of the 33 kDa protein and Ca²⁺, wasapproximately 7 electron equivalents per 200 Chl. From theseresults, it is likely that the bulky, ‘general’reductants reduce the functional Mn directly by solubilizingMn from the complex in the same way as do the water analogs.The effectiveness of these reductants in the photoactivationof the apo-water-oxidizing complex is also discussed. (Received September 13, 1989; Accepted March 12, 1990)     

Maitotoxin and P2Z/P2X7 purinergic receptor stimulation activate a common cytolytic pore

The effects ofmaitotoxin (MTX) on plasmalemma permeability are similar to thosecaused by stimulation of P2Z/P2X₇ionotropic receptors, suggesting that1) MTX directly activatesP2Z/P2X₇ receptors or2) MTX andP2Z/P2X₇ receptor stimulationactivate a common cytolytic pore. To distinguish between these twopossibilities, the effect of MTX was examined in1) THP-1 monocytic cells before andafter treatment with lipopolysaccharide and interferon-, a maneuverknown to upregulate P2Z/P2X₇receptor, 2) wild-type HEK cells andHEK cells stably expressing theP2Z/P2X₇ receptor, and3) BW5147.3 lymphoma cells, a cellline that expresses functional P2Z/P2X₇ channels that are poorlylinked to pore formation. In control THP-1 monocytes, addition of MTXproduced a biphasic increase in the cytosolic freeCa²⁺ concentration([Ca²⁺]_i);the initial increase reflects MTX-inducedCa²⁺ influx, whereas the secondphase correlates in time with the appearance of large pores and theuptake of ethidium. MTX produced comparable increases in[Ca²⁺]_iand ethidium uptake in THP-1 monocytes overexpressing theP2Z/P2X₇ receptor. In bothwild-type HEK and HEK cells stably expressing theP2Z/P2X₇ receptor, MTX-inducedincreases in[Ca²⁺]_iand ethidium uptake were virtually identical. The response of BW5147.3cells to concentrations of MTX that produced large increases in[Ca²⁺]_ihad no effect on ethidium uptake. In both THP-1 and HEK cells, MTX- andBz-ATP-induced pores activate with similar kinetics and exhibit similarsize exclusion. Last, MTX-induced pore formation, but not channelactivation, is greatly attenuated by reducing the temperature to22°C, a characteristic shared by theP2Z/P2X₇-induced pore. Together,the results demonstrate that, although MTX activates channels that aredistinct from those activated byP2Z/P2X₇ receptor stimulation, thecytolytic/oncotic pores activated by MTX- and Bz-ATP are indistinguishable.

     

Effects of light intensity and quality on the relative N and P requirement (the optimum N:P ratio) of marine planktonic algae

The relative requirement of N and P (the optimum N:P ratio)by Dunaliella tertiolecta, Phaeodactylum tricornutum, Prymnesiumparvum and Thalassiosira pseudonana was studied under variouslight intensities and spectra. The ratio was determined as theratio of the minimum cell N and P concentrations (q₀N and q₀pwhen either nutrient was limiting. The ratio varied widely amongspecies; under light-saturation for growth (116 µEin ^m–2s^–1 it ranged from 11.8 in ^D. tertiolecta to 36.6 in P.tricornutum. The ratio appeared to be higher at a sub-saturatingintensity (24 µEin m^–2 s^–1 in all except P.tricornutum, mainly because of higher q_oN with little changein q_oP. In T. pseudonana Q_oP also increased, resulting in aninsignificant change in the ratio. The ratio varied little withinthe range of saturation intensity. Light quality affected q_oNand q_oP as well as the ratio, and the pattern of change variedfrom species to species. The optimum ratio of individual specieswas linearly correlated to their q_oN except in P. tricornutum.q_oN for all species showed a linear correlation with cell proteinconcentrations irrespective of light conditions. The changeof optimum N:P ratios in the three species thus appears to berelated to changes in cell protein contents. The ratio of carbohydratesto protein remained constant regardless of light intensity orquality and was higher in P-limited cultures. We conclude thatchanges in light regime can strongly influence algal nutrientrequirements and species interrelationships by altering theoptimum cellular N:P ratio.     

Differences in nitrate and ammonia uptake among components of a phytoplankton population

We examined the NO₃^– and NH₄⁺ uptake capabilities of thedinoflagellate Peridinium cinctum and of the accompanying nanoplanktonduring the spring P. cinctum bloom in Lake Kin-neret. Throughoutthe Peridinium season, the smaller algae had greater affinitiesand faster specific uptake rates for both NO₃^–and NH₄⁺It appears that P. cinctum cannot directly compete with nanoplanktonfor nitrogen nutrients. Other factors such as the ability ofdinoflagellates to swim freely in the water column and low grazingpressures may explain their dominance in the lake.     

Dibromothymoquinone (DBMIB) Replaces the Function of QA at 77 K in the Isolated Photosystem II Reaction Center (D1-D2-Cytochrome b559) Complex: Difference Spectrum of the P680+(DBMIB-) State

Transient absorbance changes of the primary electron donor chlorophylla (P680) and acceptor pheophytin a (H) were measured at 77 Kby nanosecond laser spectroscopy in the D1-D2-cytochrome b₅₅₉photosystem II reaction center complex containing dibromomethylisopropylbenzoquinone (DBMIB). After the laser excitation of the reactioncenter in the presence of DBMIB, only the P680⁺-(DBMIB^-) statewas detected. P680⁺ mainly decayed with a t_1/e of 11 ms. Inthe absence of DBMIB, the excitation produced the P680⁺H^- radicalpair. The radical pair produced the triplet state (P680^T) witha t_1/e of 50 ns, and P680^T then decayed with a t_1/e of 2.1 ms.It was concluded that H_- was oxidized by DBMIB in a time rangefaster than the detecting time resolution (3.5 ns) even at 77K. The rapid oxidation of H^- by DBMIB was also confirmed bythe suppression of delayed fluorescence with a decay t_1/e of50 ns. The P680⁺(DBMIB^-)/P680(DBMIB) difference spectrum exhibiteda Q_y, band with a peak at 682 nm with a shoulder at 673 nm.The spectral shape was almost temperature insensitive between77 and 265 K. The feature of this spectrum in the wavelengthrange between 330 and 720 nm was compared with that of P680_T/P680or H^-/H at 77 K. (Received May 8, 1996; Accepted June 24, 1996)     

Effects of elevated CO2 concentrations on three montane grass species: III. Source leaf metabolism and whole plant carbon partitioning

Agrostis capillaris L.⁵, Festuca vivipara L. and Poaalpina L.were grown in outdoor open-top chambers at either ambient (340 3µmol mol^–1) or elevated (6804µmol mol^–1)concentrations of atmospheric carbon dioxide (CO₂) for periodsfrom 79–189 d. Photosynthetic capacity of source leaves of plants grown atboth ambient and elevated CO₂ concentrations was measured atsaturating light and 5% CO₂. Dark respiration of leaves wasmeasured using a liquid phase oxygen electrode with the buffersolution in equilibrium with air (21% O₂, 0.034% CO₂). Photo-syntheticcapacity of P. alpina was reduced by growth at 680 µmolmol^–1 CO₂ by 105 d, and that of F. vivipara was reducedat 65 d and 189 d after CO₂ enrichment began, suggesting down-regulationor acclimation. Dark respiration of successive leaf blades ofall three species was unaltered by growth at 680 relative to340 µmol mol^–1 CO₂. In F. vivipara, leaf respirationrate was markedly lower at 189 d than at either 0 d or 65 d,irrespective of growth CO₂ concentration. There was a significantlylower total non-structural carbohydrate (TNC) concentrationin the leaf blades and leaf sheaths of A. capillaris grown at680µmol mol^–1 CO₂. TNC of roots of A. capillariswas unaltered by CO₂ treatment. TNC concentration was increasedin both leaves and sheaths of P. alpina and F. vivipara after105 d and 65 d growth, respectively. A 4-fold increase in thewater-soluble fraction (fructan) in P. alpina and in all carbohydratefractions in F. vivipara accounted for the increased TNC content. In F. vivipara the relationship between leaf photosyn-theticcapacity and leaf carbohydrate concentration was such that therewas a strong positive correlation between photosynthetic capacityand total leaf N concentration (expressed on a per unit structuraldry weight basis), and total nitrogen concentration of successivemature leaves reduced with time. Multiple regression of leafphotosynthetic capacity upon leaf nitrogen and carbohydrateconcentrations further confirmed that leaf photosynthetic capacitywas mainly determined by leaf N concentration. In P. alpina,leaf photosynthetic capacity was mainly determined by leaf CHOconcentration. Thus there is evidence for down-regulation ofphotosynthetic capacity in P. alpina resulting from increasedcarbohydrate accumulation in source leaves. Leaf dark respiration and total N concentration were positivelycorrelated in P. alpina and F. vivipara. Leaf dark respirationand soluble carbohydrate concentration of source leaves werepositively correlated in A. capillaris. Changes in source leafphotosynthetic capacity and carbohydrate concentration of plantsgrown at ambient or elevated CO₂ are discussed in relation toplant growth, nutrient relations and availability of sinks forcarbon. Key words: Elevated CO₂, Climate change, grasses, carbohydrate partitioning, photosynthesis, respiration     

Induction of Glucose 6-Phosphate Transport Activity in Chloroplasts of Mesembryanthemum crystallinum by the C3-CAM Transition

To study possible changes in the transport metabolites betweenchloroplasts and cytoplasm during CAM induction of Mesembryanthemumcrystallinum, we compared substrate specificity of P1¹ translocator(s)in isolated chloroplasts from the C₃ and CAM-induced plants.The [¹⁴C]glu-cose 6-phosphate (G6P) transport activity was significantonly in the chloroplasts of CAM-mode plants and not detectablein those of C₃-mode, while a similar high rate of [³²P]P_i uptakewas observed with both types of chloroplasts. Kinetic analysisof G6P uptake in the CAM chloroplasts showed a high V_max [10.6µmol (mg Chl)^–1 h^–1] and a comparatively lowK_m value (0.41 mM); the latter was similar to K_i values of P_i,3-phosphoglycerate and phospho-enolpyruvate, 0.30, 0.34 and0.47 mM, respectively. On the other hand, [32P]Pi uptake inthe CAM chloroplasts was inhibited competitively by G6P witha K_i value (8.4 mM) 20-fold higher than the K_m value for G6Puptake, while that in C₃ chloroplasts was not inhibited at all.These results suggest that a new G6P/P_i, counterexchange mechanismis induced in the chloroplast envelope of CAM-induced M. crystallinumin addition to the ordinary type of P, translocator, that cannottransport G6P, already present in the C₃-type chloroplasts. (Received March 17, 1997; Accepted May 10, 1997)     

Reconstitution of the Photosystem I Secondary Quinone Acceptor (A1) in the P700-Fx Core Isolated from Synechococcus PCC 6301

By treating a F_A/F_B-depleted P700-F_x core from SynechococcusPCC 6301 with diethylether, most of the phylloquinone was removedwithout loss of P700. The 1 ms decay of P700⁺ in the originalcore was replaced by the 25 ns decay, which was interpretedas the backreaction occurring in a P700⁺     

Time-Resolved Spectroscopy of Chlorophyll-a like Electron Acceptor in the Reaction Center Complex of the Green Sulfur Bacterium Chlorobium tepidum

The absorption changes of chlorophyll (Chl) a-like pigments(C670) were studied by ns-ms laser spectroscopy at 77 K in theuntreated and urea-treated homodimeric reaction center (RC)complex of the green sulfur bacterium Chlorobium tepidum. Theuntreated RC complex contained 9 molecules of C670 in additionto 41 molecules of Bchl a and 0.9 molecules of menaquinone-7per one primary electron donor Bchl a dimer (P840). Upon photo-oxidationof P840, C670 showed an absorption change of a red-shift withan isosbestic wavelength at 668 nm. The absorption change ofP840 decayed with time constants (t_1/e) of 55 and 37 ms at 283and 77 K, respectively, and was assigned to represent the chargerecombination between P840⁺ and FeS^–. In the urea-treatedRC complex, a bleach peaking at 670 nm with a shoulder peakat 662 nm, which is ascribable to the reduced primary electronacceptor A₀^–, was detected after the laser excitationin addition to the shift at 668 nm indicating the formationof the P840⁺A₀^– state. The P840⁺A₀^– state decayedwith a t_1/e of 43 ns at 77 K and produced a triplet state p840^Tdue to the suppression of the forward electron transfer. Theseresults indicate the two different types of C670 species inthe RC complex; the one peaking at 670 nm functions as A⁰, whilethe other peaking at 668 nm shows the electrochromic shift,which presumably functions as the accessory pigment locatedin the close vicinity of P840. (Received May 17, 1999; Accepted July 14, 1999)     

Purification and Characterization of a Cytochrome P450 (trans-Cinnamic Acid 4-Hydroxylase) from Etiolated Mung Bean Seedlings

A Cyt P450 (P450_C4H) possessing trans-cinnamate 4-hydroxylase(C4H) activity was purified to apparent homogeneity from microsomesof etiolated mung bean seedlings. Upon SDS-polyacrylamide gelelectrophoresis, the purified preparation gave a single proteinband with a molecular mass of 58-kDa. Its specific P450 contentwas 12.6 nmol (mg protein)^–1. Using NADPH as electrondonor, purified P450_C4H aerobically converted trans-cinnamicacid to p-coumaric acid with a specific activity of 68 nmolmin^–1 nmol^–1 P450 in a reconstituted system containingNADPH-Cyt P450 reductase purified from the seedlings or rabbitliver microsomes, dilauroyl phosphatidylcholine, and cholate.This specific activity is by far the highest for reconstitutedC4H systems so far reported and provides direct evidence thatC4H activity is actually associated with a P450 protein. Inthe oxidized state P450_C4H showed a typical low-spin type absorptionspectrum with a Soret peak at 419 nm. A partial spectral shiftto the high spin state was observed when trans-cinnamic acidwas added to oxidized P450_C4H. By spectral titration, the dissociationconstant of the cinnamic acid-P450_C4H complex was determinedto be 2.8 µM. This value is similar to the K_m value (1.8µM) for trans-cinnamic acid determined in the reconstitutedsystem. (Received November 20, 1992; Accepted February 17, 1993)     

Panicum milioides, a Gramineae plant having Kranz leaf anatomy without C4-photosynthesis

Light and electron microscopic observations of the leaf tissueof Panicum milioides showed that the bundle sheath cells containeda substantial number of chloroplasts and other organelles. Theradial arrangement of chlorenchymatous bundle sheath cells,designated as Kranz leaf anatomy, has been considered to bespecific to C₄ plants. However, photosynthetic ¹⁴CO₂ fixationand ¹⁴CO₂ pulse-and-chase experiments revealed that the reductivepentosephosphate pathway was the main route operating in leavesof P. milioides. The interveinal distance of the leaves wasintermediate between C₃and C₄Gramineae species. These resultsindicate that P. milioides is a natural plant species havingchracteristics intermediate between C₃ and C₄ types. (Received March 6, 1975; )     

Effects of elevated carbon dioxide on three grass species from montane pasture II. Nutrient uptake, allocation and efficiency of use

Agrostis capillaris L.⁴ Festuca vivipara L. and Poa alpinaL.were grown in outdoor open-top chambers at either ambient (340µmol mol^–1) or elevated (680 µmol^–1)CO² for periods from 79 to 189 d. Under these conditions thereis increased growth of A. caplllarls and P. alpina, but reducedgrowth of F. vivipara. Nutrient use efficiency, nutrient productivity(total plant dry weight gain per unit of nutrient) and nutrientallocation of all three grass species were measured in an attemptto understand their individual growth responses further andto determine whether altered nutrient-use efficiencies and productivitiesenable plants exposed to an elevated atmospheric CO₂ environmentto overcome potential limitations to growth imposed by soilfertility. Total uptake of nutrients was, in general, greater in plantsof A. capillaris and P. alpina (with the exception of N andK in the latter) when grown at 680 µmol mol^–1 CO₂.In F. vivipara, however, uptake was considerably reduced inplants grown at the higher CO₂ concentration. Overall, a doubling of atmospheric CO₂ concentration had littleeffect on the nutrient use efficiency or productivity of A.capillaris. Reductions in tissue nutrient content resulted fromincreased plant growth and not altered nutrient use efficiency.In P. alpina, potassium, magnesium and calcium productivitieswere significantly reduced and photosynthetic nitrogen and phosphorususe efficiencies were doubled at elevated CO₂ with respect toplants grown at ambient CO₂ F. vivipara grown for 189 d showedthe most marked changes in nutrient use efficiency and nutrientproductivity (on an extracted dry weight basis) when grown atelevated CO₂, F. vivipara grown at elevated CO₂ however, showedlarge increases in the ratio of non-structural carbohydrateto nitrogen content of leaves and reproductive tissues, indicatinga substantial imbalance between the production and utilizationof assimilate. Key words: Nutrient, allocation, nutrient use efficiency, grasses, nutrient productivity, elevated CO₂, cliniate change     

Phosphate and Membrane Electropotentials in Trifolium repens L.

In Trifolium repens L. there were immediate transient depolarizationsof the membrane electropotential (E_vo) when KH₂PO₄ was addedto phosphate-free media, but these were of the same magnitudeas the controls (K²SO⁴ and KCI). Furthermore, the extents ofdepolarization were the same as the expected effect of the addedK⁺ calculated using the Goldman equation. There was no significantdepolarization on adding H₃PO₄ to buffered media. Consequently,there was no evidence for a depolarization caused by phosphate.This result provides evidence that the H⁺–H₂PO₄ symportin roots of T. repens operates with a stoichiometry of 1: 1. In a group of control plants ( + P plants) and a group whichwere stressed by reducing the supply of phosphate (– Pplants), the – P plants had lower values for E_vo than+P plants (– 118 mV and – 130 mV, respectively).The absence of phosphate from the measurement media also reducedE_vo (mean effect = 9 mV). A significant difference in E_vo between– P and + P plants persisted when phosphate was addedto – P plants. The electropotential difference acrossthe tonoplast (E_vo) in – P plants became more positivewith time. Key words: White clover, membrane transport, roots, tonoplast, symport     

影响叶螨磷酸酯酶活性的四因子数学模型

应用二次回归通用旋转组合设计,组建了影响叶螨磷酸酯酶（酸性和碱性）活性的四因子（缓冲液Ph值X₁、温浴时间X₂、反应温度X₃、底物浓度X₄）数学模型: Y_酸性=0.456380+0.107889X₂+0.069027X₃-0.026836X₁²-0.030794X₃², F=24.98,P<0.01;Y_碱性=0.267286-0.200736X₁+0.049541X₂+0.030930X₃-.049063X₁X₂+0.053585X₁²-0.049665X₂^2, F=57.68,P<0.01。结果表明,温浴时间是影响叶螨酸性磷酸酯酶活性的关键因子,在缓冲液pH 4.4、底物浓度8.5×10^-3 mol/L、42℃温浴40 min测得该酶活性最强。影响碱性磷酸酯酶活性的关键因子则是缓冲液pH值,pH 9.0、37℃恒温30 min、底物7.5×10^-3 mol/L的条件下,光密度值最大。两种酶的最大吸收峰波长为405 nm。     

Long-term effects of a doubled atmospheric CO2 concentration on the CAM species Agave deserti

To examine the effects of a doubled atmospheric CO₂ concentrationand other aspects of global climate change on a common CAM speciesnative to the Sonoran Desert, Agave deserti was grown under370 and 750 µmol CO₂ mol^–1 air and gas exchangewas measured under various environmental conditions. Doublingthe CO₂ concentration increased daily net CO₂ uptake by 49%throughout the 17 months and decreased daily transpiration by24%, leading to a 110% increase in water-use efficiency. Underthe doubled CO₂ concentration, the activity of ribulose-1,5-bisphosphatecarboxylase/oxygenase (Rubisco) was 11% lower, phosphoenolpyruvatecarboxylase was 34% lower, and the activated:total ratio forRubisco was 25% greater than under the current CO₂ concentration.Less leaf epicuticular wax occurred on plants under the doubledCO₂ concentration, which decreased the reflectance of photosyntheticphoton flux (PPF); the chlorophyll content per unit leaf areawas also less. The enhancement of daily net CO₂ uptake by doublingthe CO₂ concentration increased when the PPF was decreased below25 mol m^–2 d^–1 when water was withheld, and whenday/night temperatures were below 17/12 C. More leaves, eachwith a greater surface area, were produced per plant under thedoubled CO₂ concentration. The combination of increased totalleaf surface area and increased daily net CO₂ uptake led toan 88% stimulation of dry mass accumulation under the doubledCO₂ concentration. A rising atmospheric CO₂ concentration, togetherwith accompanying changes in temperature, precipitation, andPPF, should increase growth and productivity of native populationsof A. deserti. Key words: Crassulacean acid metabolism, gas exchange, global climate change, Sonoran Desert     

Nucleotide regulation of the voltage-dependent nonselective cation conductance in murine colonic myocytes

ATP is proposed to be a major inhibitory neurotransmitter in the gastrointestinal (GI) tract, causing hyperpolarization and smooth muscle relaxation. ATP activates small-conductance Ca²⁺-activated K⁺ channels that are involved in setting the resting membrane potential and causing inhibitory junction potentials. No reports are available examining the effects of ATP on voltage-dependent inward currents in GI smooth muscle cells. We previously reported two types of voltage-dependent inward currents in murine proximal colonic myocytes: a low-threshold voltage-activated, nonselective cation current (I_VNSCC) and a relatively high-threshold voltage-activated (L-type) Ca²⁺ current (I_L). Here we have investigated the effects of ATP on these currents. External application of ATP (1 mM) did not affect I_VNSCC or I_L in dialyzed cells. ATP (1 mM) increased I_VNSCC and decreased I_L in the perforated whole-cell configuration. UTP and UDP (1 mM) were more potent than ATP on I_VNSCC. ADP decreased I_L but had no effect on I_VNSCC. The order of effectiveness was UTP = UDP > ATP > ADP. These effects were not blocked by pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS), but the phospholipase C inhibitor U-73122 reversed the effects of ATP on I_VNSCC. ATP stimulation of I_VNSCC was also reversed by protein kinase C (PKC) inhibitors chelerythrine chloride or bisindolylmaleimide I. Phorbol 12,13-dibutyrate mimicked the effects of ATP. RT-PCR showed that P2Y₄ is expressed by murine colonic myocytes, and this receptor is relatively insensitive to PPADS. Our data suggest that ATP activates I_VNSCC and depresses I_L via binding of P2Y₄ receptors and stimulation of the phospholipase C/PKC pathway. inhibitory junction potentials; smooth muscle; enteric nervous system