Intracellular pH Regulation in the Giant-Celled Marine Alga Chaetomorpha darwinii

The vacuolar pH (pH_v) and the cytoplasmic pH (pH_c) of the marinegiant-celled green alga Chaetomorpha darwinii were measuredby pH microelectrode techniques on extracted vacuolar sap, andby the [^I4C]DMO distribution method respectively. Equilibrationof DMO occurred with a half-time of about 2 h, with an apparentP_DMO of 3.6 x 10^–5 cm s^–1, but the vacuolar concentrationof free, undissociated DMO was always less than the externalconcentration. The explanation offered for freshwater giant-celledalgae of net DMO^– leakage across the plasmalemma cannotapply to Chaetomorpha darwinii, since electrically-driven DMO^–exit from the cytoplasm should be similar across the plasmalemmaand the tonoplast in these cells with large, vacuole-positivepotential differences across the tonoplast. pHc was accordinglycomputed assuming either tonoplast or plasmalemma equilibrationof DMO, with correction for DMO metabolism within the cell.pH_c was 8.0–8.3 in the light in artificial seawater (pH_oabout 8.0), was some 0.5 units lower in the dark, and was slightlylower with an external pH of 7. Vacuolar pH was 6.5–6.9,without consistent effects of illumination or of external pHof 7 rather than 8. While µ_H+ at the tonoplast was similarto that in giant-celled freshwater algae (although with a greatercontribution from relative to pH), µ_H+ at the plasmalemmawas less than 8 kJ mol^–1, i.e. less than one-third ofthe value in freshwater green algae. µ_Na+ was some 13kJ mol^–1 at the plasmalemma. The possibility that theprimary active transport process at the plasmalemma of Chaetomorphadarwinii (and certain other marine algae) is Na⁺ efflux ratherthan H⁺ efflux is discussed.     

Transient Cytoplasmic pH Changes in Correlation with Opening of Potassium Channels in Eremosphaera

Steigner, W. Khler, K., Simonis, W. and Urbach, W. 1988. Transientcytoplasmic pH changes in correlation with opening of potassiumchannels in Eremosphaera.—J. exp. Bot. 39: 23–36. The role of the cytoplasmic pH (pH_c) of Eremosphaera viridisin the signal transduction chain after light-off from the chloroplaststo the K⁺ channels in the plasmalemma of this unicellular algawas investigated. The temporary opening of K⁺ channels is indicatedby a transient hypcrpolarization (TP). To record rapid changesof pH_c, continuous measurements with pH sensitive micro-electrodeswere carried out. (i) Under normal conditions pH_c in the light(7·56 ±0·2) did not differ from pHc inthe dark (7·62 ±0·2). (ii) The vacuolepH ranged between 4·8 and 5·2. (iii) After light-offa rapid transient acidification of pHc O19±0·07occurred and a TP was released, (iv) In every case, the startof the transient acidification after light-off preceded thehyperpolarization by about 3s. (v) Light-on caused a rapid transientalkalinization but never a TP. (vi) Change to acid externalmedium (3.2) transiently acidified the cytoplasm and was ableto release a TP. (vii) After addition of NH₄Cl, pH_c again showeda rapid transient acidification and the release of a TP. The origin of the protons appearing in the cytoplasm after light-offis discussed critically with respect to the buffer capacity.Either direct or indirect translocation is a possible mechanismfor the movement of H⁺ from the chloroplasts into the cytoplasm.The intracellular acidification and its relation to the openingof potassium channels in the plasmalemma leads us to suggestthat a sudden change of pH_c is a potent internal signal factorin Eremosphaera viridis. Key words: Cytoplasmic pH, transient potential, K⁺–channels, Eremosphaera viridis     

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; )     

Ammonia Uptake in the Alkalophilic Cyanobacterium Spirulina platensis

Ammonia uptake was studied in the alkalophilic cyanobacteriumSpirulina platensis. In continuous cultures under optimal growthconditions ammonia supported optimal growth (doubling time of9.3 h), causing a reduction of glutamine synthetase activityto 25% of that found in cultures grown on NO₃. Long term (20min) ammonia uptake assays were performed to study the dependencyon metabolism: 1) Ammonia uptake proceeded at the same ratesin the light and in the dark, the pH dependency pattern correlatingwith light-dependent O₂ evolution and dark O₂ consumption. 2)The uptake of ammonia was pH dependent with an optimum at pH9.3. 3) The uptake was totally dependent upon the activity ofglutamine synthetase and was completely inhibited by methoininesulfoximine. To study the mechanism by which NH₄⁺/NH₃ enters the cells, shortterm experiments (up to 1 min) were performed at pH 7.0 andpH 10.0: At pH 7.0 the uptake was slow and at a constant rate.At pH 10.0, the uptake did not saturate even at 1 mM ammoniaand the kinetics were biphasic, consisting of a fast componentlasting less than 5 seconds and of a subsequent slower component.The fast phase was insensitive to methionine sulfoximine, whereasthe slower phase was completely inhibited by this compound.We suggest that under optimal (alkaline) pH the entry of ammoniainto Spirulina cells is likely to be a pH driven diffusion process,continuously supported by its intracellular assimilation. ¹Contribution number 35 of the Microalgal Biotechnology Laboratory. (Received September 19, 1988; Accepted January 16, 1989)     

Phosphoproteins and Protein Kinase Activities Intrinsic to Inner Membranes of Potato Tuber Mitochondria

Inside-out submitochondrial particles (IO-SMP) were isolatedand purified from potato (Solanum tuberosum L. cv.) tubers.When these IO-SMP were incubated with [ ³²P]ATP more then 20proteins became labelled as a result of phosphorylation. The³²P incorporation was stimulated by the oxidising reagent ferricyanide.Except for a 17 kDa protein which was phosphorylated only inthe absence of divalent cations, the protein phosphorylationrequired Mg²⁺. The time for half-maximum ³²P incorporation was4 mm for the 22 kDa phospho-F₁ -subunit and 2 min for the 28kDa phospho-F₀ b-subunit of the proton-ATPase. The K_m for ATPfor the detected phosphoproteins was between 65 µM and110 µM. The pH optimum for protein phosphorylation ininner membranes was between pH 6 and 8, and for the F₁ -subunitand the F₀ b-subunit the pH optima were 6.5–8 and pH 8,respectively. A 37 kDa phosphoprotein was phosphorylated ona histidine residue while the remainder of the inner membraneproteins were phosphorylated on serine or threonine residues.Two autophosphorylated putative kinases were identified: oneat 16.5 kDa required divalent cations for autophosphorylation,while another at 30 kDa did not. A 110 kDa protein was labelledonly with [-³²P] suggesting adenylylation. ³ Present address; Novartis Seeds AB, Box 302, S-261 23 Landskrona,Sweden.     

Leaf Anatomy and Carbon Discrimination in NAD-malic Enzyme Panicum Species and their Hybrids Differing in Bundle Sheath Cell Ultrastructure

The relationship between leaf anatomy, ultrastructure and carbondiscrimination was investigated in leaves of two F₁hybrids (F₁-1and F₁-2) between two different types of the grassPanicum [anNAD-malic enzyme (ME) C₄species], which differ in bundle sheathultrastructure. The female parent was Kabulabula grass, whichhas centrifugal chloroplasts in bundle sheath cells and is designatedan NAD-ME(F) species, while the male parent was Makarikari grass,which has centripetal chloroplasts in the bundle sheath cellsand is designated an NAD-ME(P) species. Suberin lamellae arepresent in Kabulabula grass but are lacking in Makarikari grass.Both F₁hybrids had the same chromosome number (2n =36) as theparents but exhibited both univalent (about 45%) and bivalent(about 55%) chromosome pairing which was the major basis forthe identification of F₁hybrids. In F₁-1, elongated bundle sheathcell chloroplasts are arranged mainly in a centripetal position,similar to those in the male parent, Makarikari grass. In contrast,most of the bundle sheath cells in F₁-2 are packed with starch-containingchloroplasts, although in some cells chloroplasts tended tobe centripetally arranged. In both F₁hybrids, suberin lamellaewere found in the bundle sheath cell walls, similar to the femaleparent, Kabulabula grass. The ¹³C values of both F₁hybrids were-11.4 to -11.7, almost the same as those of Kabulabula grass(-11.4), but significantly higher than those of Makarikari grass(-12.7). These results indicate that the chloroplast orientationin the bundle sheath cells and the presence of suberin lamellaeare not obligatorily linked in their expression and suggestthat suberin lamellae may play an important role in discriminationagainst¹³C. Panicum ; NAD-malic enzyme species; hybrid; chloroplast position; ¹³C discrimination; suberin lamellae     

Pyruvate Uptake by Mesophyll and Bundle Sheath Chloroplasts of a C4 Plant, Panicum miliaceum L.

Intact chloroplasts were isolated from mesophyll and bundlesheath protoplasts of a C₄ plant, Panicum miliaceum L., to measurethe uptake of [1-¹⁴C]pyruvate into their sorbitol-impermeablespaces at 4?C by the silicone oil filtering centrifugation method.When incubated in the dark, both chloroplasts showed similarslow kinetics of pyruvate uptake, and the equilibrium internalconcentrations were almost equal to the external levels. Whenincubated in the light, only mesophyll chloroplasts showed remarkableenhancement of the uptake, the internal concentration reaching10–30 times of the external level after 5 min incubation.The initial uptake rate of the mesophyll chloroplasts was enhancedabout ten fold by light and was saturated with increasing pyruvateconcentration; K_m and V_max were 0.2–0.4 mM and 20–40µmol(mg Chl)^–1 h^–1, respectively. The lightenhancement was abolished by DCMU and uncoupling reagents suchas carbonylcyanide-m-chlorophenylhydrazone and nigericin. Theseresults indicate the existence of a light-dependent pyruvatetransport system in the envelope of mesophyll chloroplasts ofP. miliaceum. The uptake activity of mesophyll chloroplastsboth in the light and the dark was inhibited by sulfhydryl reagentssuch as mersalyl and p-chloromercuriphenylsulfonate, but thebundle sheath activity was insensitive to the reagents. Thesefindings are further evidence for the differentiation of mesophylland bundle sheath chloroplasts of a C₄ plant with respect tometabolite transport. (Received July 3, 1986; Accepted October 8, 1986)     

Studies on cytochromes in photosynthetic electron transport system I. photoreduction and photo-oxidation of cytochrome b559 by photosystem II in spinach chloroplasts

Light-induced redox-reactions of cytochrome b₅₅₉ in spinachchloroplasts were investigated. Illumination of chloroplastsinduced photoreduction of cytochrorne b₅₅₉ Red light (650 nm)was more effective than far-red light (725 nm), indicating thatthe photoreduction is a photosystem II-mediated reaction. Onaddition of DCMU, the photoreduction was eliminated and a photooxidationof cytochrome b₅₅₉ was observed. The rate of this photooxidationwas faster with photosystem II light than with photo-systemI light. On addition of Mn⁺⁺ the photooxidation was partly suppressed;far-red light became as effective as red light in inducing photooxidationof cytochrome b₅₉₉, in the presence of DCMU and Mn⁺⁺. Ascorbate completely suppressed photooxidation of cytochromeb⁵⁵⁹ In the presence of ascorbate, however, photooxidation wasobserved in the presence of inhibitors or after inhibitory treatmentsof chloroplasts which affected the oxidizing side of systemII. These inhibitors and inhibitory treatments, but not DCMU,decreased the redoxpotential of cytochrome b₅₅₉. Reactivationof Hill reaction in Tris-washed chloroplasts by indophenol-ascorbatetreatment was not accompanied by an abolishment of photooxidationof cytochrome b₅₅₉. A possible mechanism is proposed to account for these reactionsof cytochrome b₅₅₉ in the photosynthetic electron transportin chloroplasts. (Received April 4, 1972; )     

Cytoplasmic Alkalization and Cytoplasmic Streaming Induced by Light and Histidine in Leaf Cells of Egeria densa: in vivo 31P-NMR study

Rotational streaming of the cytoplasm including chloroplastswas induced by L-histidine, as well as by light, on the anticlinalface of leaf cells of Egeria densa. In the case of treatmentwith L-histidine some of the chloroplasts remained stationaryon the periclinal face of cells after rotational cytoplasmicstreaming was initiated. However, these chloroplasts were easilydislodged and translocated to the centrifugal end of the histidine-treatedcells by application of a centrifugal force that barely affectedthe location of chloroplasts in cells incubated in the darkwithout L-histidine. This result indicates that the anchoringof chloroplasts was weakened by L-histidine. Thus only the releaseof chloroplasts from anchoring was not enough for initiationof their streaming. The cytoplasmic pH (pH_c) and vacuolar pH(pH_v) were noninvasively monitored by in vivo ³¹P-nuclear magneticresonance (NMR) spectroscopy. Compared with the dark controlvalue, both illumination and treatment with L-histidine increasedthe pH_c by 0.3 units. In contrast, pH_v changed only a littlewith both illumination and treatment with L-histidine. Releaseof chloroplasts from anchoring and initiation of cytoplasmicstreaming are discussed in relation to the increase in pH_c inducedby both light and L-histidine. ⁴ Present address: Department of Cell Biology, National Instituteof Agrobiological Resources, Kannondai, Tsukuba, Ibaraki, 305Japan ⁵ Present address: Marine Biotechnology Institute Co., Ltd.,Head Office, 2-35-10 Hongo, Bunkyo-ku, Tokyo, 113 Japan (Received July 16, 1990; Accepted December 20, 1990)     

Deciphering PiT transport kinetics and substrate specificity using electrophysiology and flux measurements

Members of the SLC20 family or type III Na⁺-coupled P_i cotransporters (PiT-1, PiT-2) are ubiquitously expressed in mammalian tissue and are thought to perform a housekeeping function for intracellular P_i homeostasis. Previous studies have shown that PiT-1 and PiT-2 mediate electrogenic P_i cotransport when expressed in Xenopus oocytes, but only limited kinetic characterizations were made. To address this shortcoming, we performed a detailed analysis of SLC20 transport function. Three SLC20 clones (Xenopus PiT-1, human PiT-1, and human PiT-2) were expressed in Xenopus oocytes. Each clone gave robust Na⁺-dependent ³²P_i uptake, but only Xenopus PiT-1 showed sufficient activity for complete kinetic characterization by using two-electrode voltage clamp and radionuclide uptake. Transport activity was also documented with Li⁺ substituted for Na⁺. The dependence of the P_i-induced current on P_i concentration was Michaelian, and the dependence on Na⁺ concentration indicated weak cooperativity. The dependence on external pH was unique: the apparent P_i affinity constant showed a minimum in the pH range 6.2–6.8 of 0.05 mM and increased to 0.2 mM at pH 5.0 and pH 8.0. Xenopus PiT-1 stoichiometry was determined by dual ²²Na-³²P_i uptake and suggested a 2:1 Na⁺:P_i stoichiometry. A correlation of ³²P_i uptake and net charge movement indicated one charge translocation per P_i. Changes in oocyte surface pH were consistent with transport of monovalent P_i. On the basis of the kinetics of substrate interdependence, we propose an ordered binding scheme of Na⁺:H₂PO₄^–:Na⁺. Significantly, in contrast to type II Na⁺-P_i cotransporters, the transport inhibitor phosphonoformic acid did not inhibit PiT-1 or PiT-2 activity. Na⁺-P_i cotransport; two-electrode voltage clamp; surface pH electrode; SLC20; retroviral receptor     

Induction and Repression of CAM in Sedum telephium L. in Response to Photoperiod and Water Stress

Lee, H. S. J. and Griffiths, H. 1987. Induction and repressionof CAM in Sedurn relephluni L. in response to photopcnod andwater stress.—J. exp. Bot. 38: 834–841. The introduction and repression of CAM in Sedurn telephiunmL, a temperate succulent, was investigated in watered, progressivelydrouglited and rewatered plants in growth chambers. Measurementswere made of water vapour and CO₂ exchange, titratable acidity(TA) and xylem sap tension. Effects of photoperiod were alsostudied. CAM was induced by drought under long or short days,although when watered no CAM activity was expressed. C₃-CAM intermediate plants were used for the investigation ofwater supply. Those which had received water and those drought-stressedboth displayed a similar nocturnal increase in TA, with a day-nightmaximum (H+) of 69 µmol g^–1 fr. wt. The wateredplants took up CO₂ at a maximum rate of 2?2 µmol m^–2s^–1 only in the light period, while the droughted plantsshowed a maximum nocturnal CO₂ uptake rate of 0?69 µmolm^–2 s^–1. Subsequently, as CAM was repressed, thewatered S. telephiwn displayed little variation in TA, withconstant levels at 42 µmol g^–1 fr. wt. (day 10).After 10 d of drought stress, the CAM characteristics of S.telephiurn were aLso affected, with reduced net CO₂ uptake andH+. The transition between C₃ and CAM in S. telephium can be describedas a progression in terms of the proportion of respiratory CO₂which is recycled and refixed at night as malic acid, in comparisonwith net CO₂ uptake. Recycling increased from 20% (day 1) to44% (day 10) as a result of the drought stress and was highin both the CAM-C₃ stage (no net CO2 uptake at night) and alsoin the drought-stressed CAM stage (reduced net CO₂ uptake atnight). The complete C₃-CAM transition occurred in less than8 d, and the stages could be characterized by xylem sap tensionmeasurements: CAM = 0?50 MPa C₃-CAM = 0?36 MPa C₃ = 0?29 MPa. Key words: CAM, Sedum telephium L., recycling     

Vieillissement de l'appareil photosynth?tique II. Effet synergique de la lumi?re et du vieillissement in vitro sur les activit?es photochimiques de chloroplastes isol?s d'?pinard

The consequences of chloroplast ageing in vitro were furtherinvestigated, especially on the photochemical activities ofthese organelles. Ageing of chloroplasts in dark was accompanied by decreasesin activities for photohydrolysis and cyclic and non-cyclicsyntheses of ATP, photoreduction of NADP⁺ and O₂ evolution;but there was no decrease in ferricyanide photoreduction. Therates of decrease in these activities were comparable to therate of increase in chloroplast volume. Complete inhibitionswere reached when maximum chloroplast swelling had occurred,i.e. after 5 to 6 hr of incubation at 20?C in a Tris-NaCl (pH8) medium. Ageing in the light resulted in much accelerateddecreases in activities tested; the loss of capacity for light-inducedshrinkage was also accelerated by the light during ageing. Thus,light acts synergetically towards the ageing process. Moreover,light and, to a less extent, dark ageing, resulted in an uncouplingof chloroplast photophosphorylation and associated electronflow measured by ferricyanide photoreduction. The part of theelectron flow which is induced by coupling (+ ADP, Pi, MgCl₂,pH 8) or by uncoupling (+ NH₄C1, pH 7) was found to be verysensitive to light ageing. The NADP⁺ photoreduction loss wasrestored by addition of the ascorbate-DCPIP electron donor system,suggesting that ageing interferes with the integrity of photosystemII. In many respects, these effects of ageing are comparable withthe action of detergents and fatty acids on the structure andphotochemical activities of chloroplasts, especially in thatthey attack the energy transducing mechanism in chloroplasts. (Received May 24, 1969; )     

Effect of Low Relative Humidity on {delta}13C Value in Two C3 Grasses and in Panicum milioides, a C3-C4 Intermediate Species

When grown under conditions of low relative humidity, the C₃–C₄intermediate Panicum milioides, as well as the C₃ grasses Triticumaestivum and Poa pratense, exhibited ¹³C values which were upto 2–7%o less negative than the ¹³C values of the correspondingplants grown at high relative humidity. At both humidity levels,there was no evidence of a substantial contribution of phosphoenolpyruvatecarboxylase to carbon gain in Panicum milioides     

Light-induced ATP Formation from Acetyl Phosphate and ADP by Broken Spinach Chloroplasts

Spinach chloroplasts catalyzed ATP formation from acetyl phosphateand ADP when exposed to light. No ATP formation was detectablein the dark. In the absence of ADP, chloroplasts did not hydrolyzeacetyl phosphate in the light or dark. Neither high-energy phosphatessuch as creatine phosphate and phosphoenol pyruvate nor inhibitorsof photophosphorylation competitive with P_i, such as ß-naphthylmonophosphate, phenyl phosphate and pyridoxal 5-phosphate, couldsubstitute for acetyl phosphate as a P_i donor. The apparentK_m values for acetyl phosphate and P_i were 0.81 mM and 0.25mM, respectively. The maximal rate of ATP formation with acetylphosphate and P_i were 331 and 521 µmol ATP formed mg chl^–1hr^–1, respectively. The optimum pH value for acetyl phosphate-dependentATP formation was about 8.0. NH₄Cl, dicyclohexylcarbodiimideand triphenyltin chloride inhibited the acetyl phosphate-dependentATP formation. Acid-base transition also could induce subsequentATP formation from acetyl phosphate and ADP. These results suggestthat the acetyl phosphate-dependent ATP formation requires theformation and the utilization of a proton-motive force as ordinaryphotophosphorylation does. ¹ This work was supported in part by Grants-in-Aid for ScientificResearch from the Ministry of Education, Science and Culture,Japan to H. S. Part of this work was reported at the 1981 AnnualMeeting of the Japanese Society of Plant Physiologists (Sapporo,May 8, 1981). (Received August 25, 1981; Accepted November 1, 1981)     

Induction of Light Dependent Pyruvate Transport into Chloroplasts of Mesembryanthemum crystallinum by Salt Stress

Mode of photosynthesis in Mesembryanthemum crystallinum changesfrom C₃ to Crassulacean acid metabolism (CAM) when the plantswere stressed with high salinity. [¹⁴C]Pyruvate uptake for 30s into intact chloroplasts isolated from leaves of the CAM modeof M. crystallinum was enhanced more than 5-fold in the lightcompared with that in the dark. The stromal concentration ofpyruvate in the light reached to more than 2.5 times of themedium. In contrast, little or no pyruvate uptake occurred inchloroplasts from C₃ leaves in either light or dark condition.The initial uptake rate (10 s incubation at 4°C) into theCAM chloroplasts in the light was about 3-fold higher than therate in the dark. K_m and V_max of the initial uptake in the lightwere 0.54 mM and 8.5 µmol (mg Chl)^–1 h^–1 respectively.These suggest that pyruvate was actively incorporated into theCAM chloroplasts against its concentration gradient across theenvelope in the light. When hydroponically grown M. crystallinumwere stressed by 350 mM NaCl, the capacity of chloroplasts forpyruvate uptake was induced in 6 d corresponding to the inductionof the activities of PEP-carboxylase and NAD(P)⁺-malic enzymesin response to salt stress. (Received October 12, 1995; Accepted January 19, 1996)     

Temporal variations in carbon isotope ratio of phytoplankton in a eutrophic lake

Temporal variations in carbon isotope ratio of phytoplanktonand dissolved inorganic carbon (DIC) in Lake Suwa were reported.In summer, blooming of Microcystis spp. resulted in low concentrationsof DIC and high pH, and HCO₃^– was the prominent speciesof DIC. Chlorophyll-specific rates of photosynthesis were relativelyconstant irrespective of the algal biomass during summer. Carboxylationin photosynthesis of Microcystis spp. was mainly catalyzed byribulose bisphosphate carboxylase (RuBPCase). Carbon isotopediscrimination between ¹³C of phytoplankton and DIC was considerablysmall in early summer and appeared to be negatively correlatedto DIC concentration. We concluded that carbon fixation by phytoplanktonin Lake Suwa is controlled not by the switch of photosyntheticpathways, but by low DIC concentration and high pH, suggestingthat photosynthesis of Microcystis spp. in Lake Suwa is governedby uptake kinetics other than the carboxylation step.     

Proton/Pyruvate Cotransport into Mesophyll Chloroplasts of C4 Plants

Light-enhanced active pyruvate uptake into mesophyll chloroplastsof C₄ plants was reported to be mimicked by either of the twotypes of cation jump: H⁺-jump in maize and phylogenically relatedspecies (H⁺-type) and Na⁺-jump in all the other C₄ species tested(Na⁺-type) [Aoki, N., Ohnishi, J. and Kanai, R. (1992) PlantCell Physiol. 33: 805]. In this study, medium and stromal pH was monitored in the suspensionof C₄ mesophyll chloroplasts. Medium alkalization lasting for5 to 10 seconds after pyruvate addition was detected by a pHelectrode and observed only in the light and only in mesophyllchloroplasts from H⁺-type species, Zea mays L. and Coix lacryma-jobiL., but not in those from Na⁺-type species Panicum miliaceumL., Setaria italica (L.) Beauv. and Panicum maximum Jacq. Theinitial rate of H⁺ consumption showed good correlation with[¹⁴C]pyruvate uptake measured by silicone oil filtering centrifugation,both being inhibited by N-ethylmaleimide and 7-chloro-4-nitrobenzo-2-oxa-l,3-diazole to the same degree. The ratio of the rate of H⁺ uptaketo that of pyruvate uptake was always about 1. Pyruvate-inducedacidification of the stroma was observed in maize mesophyllchloroplasts. These results show one to one cotransport of H⁺and pyruvate anion into mesophyll chloroplasts of H⁺-type C₄species in the light. (Received January 5, 1994; Accepted May 6, 1994)     

Fractionation of Nitrogen Isotopes during the Uptake and Assimilation of Ammonia by Plants

Two varieties (Nihonbare and Koshihikari) of rice plants (Oryzasativa L.) were grown hydro-ponically with two levels (20 and100 mg N liter ^–1) of ammonia. Variations in levels ofnatural abundance of ¹⁵N (¹⁵N) were analyzed in the ammoniaand organic nitrogen of shoots and roots, as well as in theammonia in the culture solution. There was substantial fractionationof nitrogen isotopes during the uptake of ammonia. When plantsabsorbed a large proportion of ammonia from a solution witha low concentration, less negative ¹⁵N values in plants andhigh positive ¹⁵N values in the ammonia remaining in solutionwere observed. The reverse was found when a smaller fractionof ammonia was absorbed from a solution with a higher concentrationof ammonia. The ^l5N values of ammonia in shoots and roots werehigher than in the respective constituent organic nitrogen,suggesting the fractionation of nitrogen isotopes during theassimilation of ammonia. Wild-type and mutant cells of the cyanobacterium(blue-green alga) Synechococcus PCC 7942 were grown in nitrate-or ammonia-containing medium as the source of nitrogen. Fractionationof nitrogen isotopes during the uptake of nitrate was limited,whereas that during the uptake of ammonia was considerable. ¹ In this report, the term ammonia refers indiscriminately toboth NH₃ or NH₄⁺. (Received June 13, 1991; Accepted September 12, 1991)     

Reappraisal of the Role of Sodium in the Light-Dependent Active Transport of Pyruvate into Mesophyll Chloroplasts of C4 Plants

The mechanism of light-dependent active transport of pyruvatein C₄ mesophyll chloroplasts has not been clarified, particularlyin Na⁺-type C₄ species, in which the pyruvate uptake into mesophyllchloroplasts is enhanced by illumination or by making a Na⁺gradient (Na⁺-jump) across the envelope in the dark. We re-investigatedhere the effect of Na⁺ on the active transport of pyruvate inmesophyll chloroplasts of Panicum miliaceum, a Na⁺-type C₄ species,by comparing the rate of pyruvate uptake at various externalpHs under four conditions; in the light and dark together with/withoutNa⁺-jump: (1) At neutral pH, the rate of pyruvate uptake inthe dark was enhanced by Na⁺-jump but scarcely by illumination.(2) While the enhancement effect by Na⁺-jump was independentof external pH, that by illumination increased greatly at pHover 7.4, and the effects of light and Na⁺ at the alkaline pHwere synergistic. (3) The light-enhanced pyruvate uptake wasrelated to stromal alkalization induced by illumination. Infact, pyruvate uptake was induced by H⁺-jump in the medium frompH 8.0 to 6.7. (4) Stromal pH was lowered by the addition ofK⁺-pyruvate and more by Na⁺-pyruvate into the medium at pH 7.8in the light. (5) However, the pH and ATP levels in the stromawere not affected by Na⁺-jump. Thus, we discussed possibility that besides pyruvate/Na⁺ cotransportat neutral pH in the medium, pyruvate/H⁺ cotransport enhancedby the presence of Na+ operates in mesophyll chloroplasts ofNa⁺-type C4 species at alkaline medium. ¹Present address: Biological Resources Division, Japan InternationalResearch Center for Agricultural Sciences (JIRCAS), Ministryof Agriculture, Forestry and Fisheries, 2-1 Ohwashi, Tsukuba,305 Japan     

Channel phosphorylation and modulation of L-type Ca2+ currents by cytosolic Mg2+ concentration

Previous studies have shown that inhibition of L-type Ca²⁺ current (I_Ca) by cytosolic free Mg²⁺ concentration ([Mg²⁺]_i) is profoundly affected by activation of cAMP-dependent protein kinase pathways. To investigate the mechanism underlying this counterregulation of I_Ca, rat cardiac myocytes and tsA201 cells expressing L-type Ca²⁺ channels were whole cell voltage-clamped with patch pipettes in which [Mg²⁺] ([Mg²⁺]_p) was buffered by citrate and ATP. In tsA201 cells expressing wild-type Ca²⁺ channels (_1C/_2A/₂), increasing [Mg²⁺]_p from 0.2 mM to 1.8 mM decreased peak I_Ca by 76 ± 4.5% (n = 7). Mg²⁺-dependent modulation of I_Ca was also observed in cells loaded with ATP--S. With 0.2 mM [Mg²⁺]_p, manipulating phosphorylation conditions by pipette application of protein kinase A (PKA) or phosphatase 2A (PP_2A) produced large changes in I_Ca amplitude; however, with 1.8 mM [Mg²⁺]_p, these same manipulations had no significant effect on I_Ca. With mutant channels lacking principal PKA phosphorylation sites (_1C/S1928A/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p had only small effects on I_Ca. However, when channel open probability was increased by _1C-subunit truncation (_1C1905/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p greatly reduced peak I_Ca. Correspondingly, in myocytes voltage-clamped with pipette PP_2A to minimize channel phosphorylation, increasing [Mg²⁺]_p produced a much larger reduction in I_Ca when channel opening was promoted with BAY K8644. These data suggest that, around its physiological concentration range, cytosolic Mg²⁺ modulates the extent to which channel phosphorylation regulates I_Ca. This modulation does not necessarily involve changes in channel phosphorylation per se, but more generally appears to depend on the kinetics of gating induced by channel phosphorylation. voltage-gated Ca²⁺ channel; cardiac myocytes; human embryonic kidney cells; protein kinase A; protein phosphatase 2A