Kinetic Characterization of Two Phosphate Uptake Systems with Different Affinities in Suspension-Cultured Catharanthus roseus Protoplasts

We studied the kinetics of inorganic phosphate (P₁) uptake from0.1–1,000 µM P₁ by protoplasts from suspension-culturedcells of Catharanthus roseus (L.) G. Don. Concentration dependenceof [³²P]P₁ uptake revealed two kinetically different uptakesystems, a high-affinity system and a low-affinity system, withK_m values of 3.0 and 47 µM, respectively. Protoplastsfrom cells grown in P_i-rich media had a medium level of thelow-affinity activity and a very low level of the high-affinityactivity. It appeared low-affinity system is expressed constitutively,while the high-affinity system is regulated by the availabilityof P_i. When cells grown in a P_i-rich media were transferredto P_i-depleted media, the high-affinity activity increased significantlyafter 2 d, but the low-affinity activity was barely changed.Upon addition of 10 mM P_i, the high level of the high-affinityactivity fell to almost undetectable level in 1d. Both uptakesystems exhibited maximum activity between pH 5 and 6. ¹ Present address: Tokyo Research Laboratories, Kyowa HakkoKogyo Co., Ltd., 3-6-6 Asahi-cho, Machida, Tokyo, 194 Japan.     

Effect of Inorganic Phosphate on the Biosynthesis of Purine and Pyrimidine Nucleotides in Suspension-Cultured Cells of Catharanthus roseus

The levels of purine and pyrimidine nucleotides in suspensioncultures of Catharanthus roseus were determined 24 h after stationary-phasecells were transferred to fresh complete (‘+Pi’)or phosphate-deficient (‘–Pi’) Murashige-Skoogmedium. The levels of ATP, GTP, UTP and CTP were from approx.3 to 5-fold greater in the cells grown in ‘+Pi’medium than in the cells grown in ‘–Pi’ medium.The levels of almost all other nucleotides were slightly higherin the cells in ‘+Pi’ medium. The rates of de novoand salvage biosynthesis of purine and pyrimidine nucleotideswere estimated from the rates of incorporation of radioactivityfrom [¹⁴C]formate, [2–¹⁴C]glycine, NaH¹⁴CO₃, [6–¹⁴C]orotate,[8–¹⁴C]adenine, [8–¹⁴C]adenosine, [2–¹⁴C]uraciland [2–¹⁴C]uridine. The results indicated that the activityof both the de novo and the salvage pathway was higher in thecells in ‘+Pi’ medium than in the cells in ‘–Pi’medium. The rate of degradation estimated from the rate of releaseof ¹⁴CO₂ from labelled purines and pyrimidines indicated thatdegradation of uridine was significantly reduced in the cellsin ‘+Pi’ medium, but no significant difference wasfound in the degradation of adenine, adenosine and uracil. Thepossible role of Pi in the control of the biosynthesis of nucleotidesand in the degradation of uridine is discussed. Catharanthus roseus, Madagascar periwinkle, suspension culture, inorganic phosphate, nucleotides, purines, pyrimidines, biosynthesis, degradation     

ERRATA

Effects of coupled solute and water flow in plant roots withspecial reference to Brouwer's experiment. Edwin L. Fiscus. p. 71 Abstract: Line 3 delete ‘interval’ insert‘internal’. p. 73 Materials and Methods: line 6: delete ‘diversion’ insert ‘division’ line 9 equation should read J_v=L_p P–RT(C⁰–C¹). 74 Last line of figure legend: 10^–1 should read 10^–11. 75 Line 11: delete ‘seems’ insert ‘seem’. le 1 column heading—10⁶ should read 10¹¹. 77 delete ‘...membrane in series of...’ insert ‘membranein series or...’ Delete final paragraph.     

Effects of Inorganic Phosphate on the Utilization of Sucrose by Suspension-cultured Catharanthus roseus Cells

The metabolic fate of [U-¹⁴C]sucrose in suspension culturesof Catharanthus roseus cells was monitored for 96 h after thecells were transferred to fresh complete (‘+Pi’)or to phosphate-deficient Murashige and Skoog (‘–Pi’)medium. Sucrose was hydrolysed extracellularly to glucose andfructose. The rate of uptake of sugars by the cells was 1.5–3times higher in ‘+Pi’ culture than in ‘–Pi’culture. Little difference in the rate of incorporation of radioactivityinto the ethanol-soluble fraction was found between the ‘+Pi’and ‘– Pi’ cultures during the initial 24h of culture, but after 48 h the rate in ‘ +Pi’cultures was higher than that in ‘–Pi’ cultures.Incorporation of radioactivity into ethanol-insoluble macromoleculeswas always significantly higher in the cells in ‘+Pi’cultures than in those in ‘–Pi’ cultures.The results suggest that Pi strongly affects the utilizationof sugars by cultured plant cells through the stimulation oftransport of sugars as well as through the activation of metabolism. Catharanthus roseus, Madagascar periwinkle, suspension culture, inorganic phosphate, sucrose, transport, metabolism     

Degradation of Endogenous Organic Acids Induced by Pi Uptake in Catharanthus roseus Cells: Involvement of the Biochemical pH-Stat

We have previously shown that inorganic orthophosphate (P_i)uptake by Catharanthus roseus cells proceeds by a proton/P_icotransport mechanism [Sakano (1990) Plant Physiol. 93: 479]that acidifies the cytoplasm [Sakano et al. (1992) Plant Physiol.99: 672]. In the present study, we analyzed changes in the contentof endogenous organic acids, carbon dioxide evolution, and oxygenconsumption upon P_i application. The results are consistentwith the operation of the biochemical pH-stat mechanism [Davies(1986) Physiol. Plant. 67: 702] during and after P_i uptake. (Received November 13, 1997; Accepted March 30, 1998)     

The Apparent Induction of Nitrate Uptake by Chara corallina Cells following Pretreatment with or without Nitrate and Chlorate

Nitrate provision has been found to regulate the capacity forChara corallina cells to take up nitrate. When nitrate was suppliedto N sufficient cells maximum nitrate uptake was reached after8 h. Prolonged treatment of the cells in the absence of N alsoresulted in the apparent ability of these cells to take up nitrate.Chlorate was found to substitute partially for nitrate in the‘induction’ step. The effects on nitrate reductionwere separated from those on nitrate uptake by experiments usingtungstate. Tungstate pretreatment had no effect on NO^–₃uptake ‘induced’ by N starvation, but inhibitedNO^–₃ uptake associated with NO^–₃ pretreatment. Chloridepretreatment similarly had no effect on NO^–₃ uptake ‘induced’by N deprivation, but inhibited NO^–₃ uptake followingNO^–₃ pretreatment. The data suggest that there are atleast two mechanisms responsible for the ‘induction’of nitrate uptake by Chara cells, one associated with NO^–₃reduction and ‘induced’ by CIO^–₃ or NO^–₃and one associated with N deprivation. Key words: Nitrate, Chlorate, Chara corallina, Induction     

The Sensitivity of Net Photosynthesis in Several Plant Species to Short-term Fumigation with Sulphur Dioxide

The effects of exposure of up to 2 h with sulphur dioxide ona range of plant species was observed by measuring changes inthe rate of net photosynthesis under closely controlled environmentalconditions. Ryegrass, Lolium perenne ‘S23’ was thespecies most sensitive to SO₂; significant inhibition was detectedat 200 nl l^–1. Fumigations at 300 nl l^–1 also inhibitedphotosynthesis in field bean (Vicia faba cv. ‘Three FoldWhite’ and ‘Blaze’) and in barley (Hordeumvulgare cv. ‘Sonja’). No effect was detected inwheat (Triticum aestivum cv. ‘Virtue’) at concentrationsup to 600 nl l^–1 SO₂, or in oil-seed rape (Brassica napuscv. ‘Rafal’) except at 800 nl l^–1 SO₂). Recoverycommenced immediately after the fumigation was terminated andwas complete within 2 h when inhibition had not exceeded 20%during the SO₂ treatment. Key words: Sulphur dioxide, short-term fumigation, photosynthesis     

Endogenous Levels of Gibberellins, IAA and Cytokinins in Catharanthus Crown Gall Tissues of Different Tumor Types

Endogenous levels of gibberellins, auxins and cytokinins incrown gall tissues of Catharanthus roseus G. Don. (Vinca roseaL.) of two different tumor types, induced by a nopaline-typeand an octopine-type Ti-plasmid, were examined. The levels of gibberellins were higher in nopaline-type V208cells than in octopine-type V277 cells. GA₁₂ and GA₂₄ were identifiedfrom V208 cells. The level of IAA was 20-fold higher in V208cells than in V277 cells. The level of trans-ribosylzeatin washigher in V277 cells than in V208 cells, whereas trans-zeatinwas present at higher levels in V208 cells than in V277 cells. Our results revealed that the production of gibberellins, aswell as of IAA and cytokinins, in the crown gall tissues ofCatharanthus roseus differed between the octopine- and nopaline-typetumors, even though the crown galls apparently grow as unorganizedaggregates of cells in both cases. ⁴Present address: Facultad de Ciencias, Pontificia UniversidadJaveriana, Bogota, Colombia. (Received September 29, 1989; Accepted February 2, 1990)     

Phenylpropanoids as a Protectant of Aluminum Toxicity in Cultured Tobacco Cells

Aluminum (Al) enhances ferrous ion [Fe(II)]-mediated peroxidationof lipids, which is lethal to normal tobacco cells, but notto phosphate (P_i)-starved cells ( –P cells). We foundthat tobacco cells accumulated phenylpropanoid compounds includingchlorogenic acid (CGA) and caffeic acid (CA) during P_i starvation.The accumulation was inhibited by 2-aminoindan-2-phosphonicacid (AIP), a specific inhibitor of L-phenylalanine ammonialyase (PAL). CGA, CA and also an extract containing the phenylpropanoidcompounds from –P cells protected normal cells ( +P cells)efficiently from both lipid peroxidation and the loss of viabilitycaused by the combined application of Al and Fe(II), indicatingthat the phenylpropanoids acted as antioxidant molecules. –Pcells exhibited approximately 25-fold higher specific activityof PAL than +P cells. The content of the phenylpropanoids andthe activity of PAL were not affected by the combined treatmentwith Al and Fe(II) in either +P cells or –P cells. Theseresults suggest that an increase in PAL activity during P_i starvationenhances the accumulation of phenylpropanoids, and that thephenylpropanoids protect tobacco cells from cytotoxic lipidperoxidation caused by the combination of Al and Fe(II) ⁴CREST, Japan Science and Technology Corporation (JST).     

A Cytochrome P450 Mediated Naringenin 3'-Hydroxylase from Sweet Orange Cell Cultures

A microsomal flavonoid 3'-hydroxylase (F3'H) catalyzing themetabolism of naringenin to eriodictyol in Citrus sinensis (L.)Osbeck cv. ‘Hamlin’ cell suspension cultures wasshown to be a cytochrome P450 enzyme. This reaction requiredO₂ and NADPH and was inhibited by CO, with partial reversalof CO-inhibition by light at 450 nm. Cytochrome P450 contentranged from 10–20 pmol (mg microsomal protein)^–1.The F3'H reaction was shown to be linear in regard to proteinconcentration between 2.5 and 25 µg of microsomal protein.The optimum pH for the reaction was 7.4–7.6 and the temperatureoptimum was between 30 and 37°C. The apparent K_m and V_maxfor naringenin were 24 µM±3.2 and 81.4±7.9pmol eriodictyol min^–1 (mg protein)^–1, respectively.The microsomal F3'H was also capable of forming dihydroquercetinfrom dihydrokaempferol (40 pmol min^–1 (mg protein)^–1)and of quercetin from kaempferol (3.25 pmol min^–1 (mgprotein^–1). Cytochrome c and ketoconazole were the bestinhibitors of WH activity followed by piperonyl butoxide anda-naphthoflavone. Light was shown to be an inducer of the F3'Halmost doubling the specific activity and increasing the microsomalcytochrome P450 content by 30% over that of dark grown cells.F3'H activity was also confirmed in microsomal preparationsof young (new flush) leaves from ‘Hamlin’ treesand flavedo of ‘Hamlin’ oranges, ‘Marsh’grapefruit, and ‘Lisbon’ lemon. No activity wasobserved in older, hardened leaves and albedo of all the fruittested. Initiation of embryogenesis in the ‘Hamlin’cell suspension cultures by switching from a sucrose mediumto a glycerol-based medium resulted in the down-regulation ofF3'H. ¹Mention of a trademark, warranty, proprietary product, or vendordoes not constitute a guarantee by the U.S. Department of Agricultureand does not imply its approval to the exclusion of other productsor vendors that may also be suitable.     

Participation of Inorganic Orthophosphate in Regulation of the Ribulose-1,5-bisphosphate Carboxylase Activity in Response to Changes in the Photosynthetic Source-Sink Balance

Sink-limited conditions, defined as treatment with continuousillumination, cause a reduction in the rate of photosyntheticfixation of CO₂ in single-rooted leaves of soybean (Glycinemax. Merr. cv. Turunoko). We suggested previously that thisreduction is due to a deactivation of ribulose-1,5-bisphosphatecarboxylase (RuBPcase, E.C. 4.1.1.39 [EC] ) that is caused by a decreasein the level of P_i in the leaves [Sawada et al. (1989) PlantCell Physiol. 30: 691, Sawada et al. (1990) Plant Cell Physiol.31: 697]. In the present study, the mechanism of regulationof RuBPcase activity by P_i was examined. The activity of RuBPcasein the sink-limited leaves, exposed for 6 or 7 d to continuousillumination to alter the source/sink balance, was enhancedwith increasing concentrations of P_i, in a CO₂-free preincubationmedium in the presence of 5 mM MgCl₂ The maximum value [6.3µmole CO₂ (mg Chl)^–1 min^–1] was obtained atapproximately 5 mM P_i after a 5 min incubation, being 3 timesof the activity without the preincubation. The activity of acrude preparation of RuBPcase that had been deactivated by removalof CO₂ and Mg²⁺ ions by the gel filtration was 5.2–9.3nmole CO₂ (mg protein)^–1 min^–1 and was also enhancedby P_i plus Mg²⁺ ions. The maximum value [147–151 nmoleCO₂ (mg protein)^–1 min^–1] was attained at 5 mM P_iafter a 5 min incubation. The cycle of activation and inactivationof deactivated crude RuBPcase was perfectly reversible by additionof P_i to the enzyme and removal of P_i from the enzyme. Levelsof free P_i and of esterified phosphate in the sink-limited leaveswere 69% and 31% of the total phosphate, respectively. By contrast,in the control leaves, these values were 87% and 13%, respectively.These results support our previously stated hypothesis and indicatean important role for free P_i in the regulation of RuBPcaseactivity, in particular in sink-limited plants. (Received February 21, 1992; Accepted July 23, 1992)     

Pyridoxal 5-phosphate, Phenyl Phosphate and Acetyl Phosphate, as Inhibitors of Photophosphorylation Competitive with Phosphate

Pyridoxal 5-phosphate, phenyl phosphate and acetyl phosphate,as well as rß-naphthyl monophosphate, inhibited photophosphorylationof spinach chloroplasts competitively with P_i and noncompetitivelywith ADP. The apparent dissociation constant of the inhibitor-enzymecomplex (K_i) values of pyridoxal 5-phosphate, phenyl phosphateand acetyl phosphate for the P_i site were 1.1, 3.8 and 2.4 _mM,respectively. These organic phosphates inhibited Ca²⁺-ATPaseof the isolated coupling factor 1 (CF₁) (EC 3.6.1.3 [EC] ) noncompetitivelywith ATP. AMP, creatine phosphate, fructose 1,6-bisphosphate,glucose 6-phosphate, 3-phosphoglyceric acid, ribose 5-phosphateand PP_i did not significantly inhibit photophosphorylation.Like rß-naphthyl monophosphate, pyridoxal 5-phosphateand phenyl phosphate inhibited photophosphorylation and thecoupled electron transport, but were almost without effect onthe basal electron transport. On the other hand, acetyl phosphateconsiderably inhibited photophosphorylation, but had almostno effect on the coupled electron transport rate and the basalrate. The results suggest that these organic phosphates inhibitphotophosphorylation by binding at the P_i site on the activecenter of CF₁ and that their binding inhibits the ATPase activityof isolated CF₁. These four organic phosphates which inhibited photophosphorylationcompetitively with Pi could not substitute for ADP or ATP ininhibiting ferricyanide photoreduction by decreasing H⁺-permeabilitythrough CF₁ and in protecting the ATPase of isolated CF₁ againstcold-anion inactivation. 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. (Received May 25, 1981; Accepted September 28, 1981)     

Leaf Abscission and Stem Lesions (Intumescences) on Poplar Clones after SO2 and O3 Fumigation: A Link with Ethylene Release?

Differences in premature leaf abscission and in visible steminjury in genetic lines of poplar followed continuous fumigationswith air pollutant levels of SO₂ (90–100 nl l^–1)and O₃ (70–80 nl l^–1) either separately or together.The clones used were: Populus deltoides var. missiouriensisMarsh., P. nigra cv. ‘italicd’ L., and the hybridsP. nigra cv. ‘italica’ xP. deltoides (He-X/3) andP. nigra cv.‘italica’ x P. nigra cv. ‘Serres’(He-K/7). While most leaf abscission occurred within 20 d fromthe start of fumigation, stem lesions (intumescences), appearedonly after 72 d. Their anatomical characteristics include theformation of lysigenous aerenchyma in the lower parts of theintumescence, the sloughing of superficial cells from the injuredarea, and the development of crystalline formations on the surfaceof the lesions. P. deltoides exhibited the least morphologicalresponse to the gases. Ethylene released from fumigated leaves was determined at thesame gas concentration of SO₂ (100 nl l^–1), O₃ (75 nll^–1) and their mixture. Leaves of P. deltoides consistentlyshowed the lowest ethylene production after the gas treatments.P. ‘italica’ production was higher but was littlealtered by the treatments. The two hybrids He-X/3 and He-K/7showed the greatest increases in ethylene evolution with time.With He-K/7 exposed to the gas mixture the production of ethylenedecreased after the initial sharp rise during days 1–2,and reflected the considerable leaf damage observed after day3. The results suggest that sensitivity to air pollution, (as shownby leaf abscission and the formation of stem intumescences)can be correlated with the level of pollutant-induced ethyleneevolution from leaves. Initially high levels could induce abscission,whilst prolonged production could be responsible for intumescenceinitiation. The discussion proposes a series of events fromSO₂ and/or O₃ entry into the leaf and the physiological reasonsfor the clonal differences. Key words: Sulphur dioxide, ozone, ethylene, poplar, leaf abscission, stem lesions     

Uptake of Inorganic Phosphate by Suspension-Cultured Tobacco Cells: Kinetics and Regulation by Pi Starvation

The kinetics of P_i uptake by phosphate-starved and non-starvedtobacco cells (Nicotiana tabacum BY-2) suspension culture wasinvestigated. The kinetic parameters of P_i uptake were determinedby computer simulation of the curve that represented the time-dependentloss of P_i from the culture medium. The uptake profile couldbe completely explained by assuming the existence of only onekind of Michaelis-Menten-type P_i-transport system with an affinityfor P_i (K_m) of about 2.5 µM (the lowest value reportedto date) in both P_i-starved and non-starved cells. No evidencewas obtained suggesting the existence of a "low-affinity" P_i-uptakesystem that has been postulated to exist in several other plantmaterials. The V_max for uptake of P_i by non-starved cells was12 nmol per minute per milliliter of packed cell. Phosphatestarvation increased the V_max more than 5-fold, while it hadno effect on the affinity for P_i. V_max began to increase (atan almost constant rate) just after loss of all P_i from theculture medium and it reached a maximum about 16 hours later.This induction process was completely prevented by the additionof cycloheximide to the culture medium. All these results suggestthat P_i starvation increases the synthesis of a phosphate-carriercomplex that is postulated to be involved in the P_i-uptake process. (Received August 12, 1994; Accepted December 26, 1994)     

Furosemide: a Specific Inhibitor of Pi Transport across the Plasma Membrane of Plant Cells

A search was made for inhibitors of P_i uptake that act directlyon the P_i transporter in the plasma membranes of Catharanthusroseus cells to inhibit P_i uptake without inhibition of protonpumping. Using standard electrodes, we monitored changes inpH and in the concentration of K⁺ ions, as well as the rateof P_i uptake, when an inhibitor to be tested was applied tothe cells in unbuffered medium. A9C (28 µM), a blockerof anion channels, inhibited P_i uptake but it also inhibitedthe proton pump. However, a structurally similar inhibitor,furosemide, inhibited P_i uptake without inhibiting proton pumping. It is suggested that the carboxylic group of these inhibitorsinteracts with the P_i-binding site (probably an amino group)of the P_i transporter in the plasma membrane and that the hydrophobicstructure of these inhibitors facilitates their accumulationin the plasma membrane. ³Present address: Department of Biology, Hitotsubashi University,2-1 Naka, Kunitachi, Tokyo, 186 Japan     

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     

Regulation of Ribulose-l,5-bisphosphate Carboxylase Activity in Response to Changes in the Source/Sink Balance in Single-Rooted Soybean Leaves: The Role of Inorganic Orthophosphate in Activation of the Enzyme

The results of our previous study [Sawada et al. (1989) PlantCell Physiol. 30: 691] implied that, under sink-limited conditions,a decrease in the activity of ribulose-l,5-bisphosphate carboxylase(EC 4.1.1.39 [EC] ) caused a reduction in the rate of photosyntheticfixation of CO₂ in single-rooted leaves of soybean (Glycinemax L. Merr. cv. Tsurunoko). This reduction in the rate of photosynthesisin source leaves seemed to correspond to a decrease in the demandby sink tissues for photoassimilates. In the present study,the activity of RuBPcase in vivo was estimated by measuringthe "initial" activity immediately after extraction from standardleaves (grown under a regime of 10 h of light and 14 h of darkness)and from sink-limited leaves (exposed for 6 or 7 d to continuouslight to alter the source/sink balance). The rate of photosynthesisin the sink-limited leaves decreased to 47% of that in the standardleaves. The "initial" activity of RuBPcase was 4.3 in the standardleaves but only 1.6 µmole CO₂.(mg Chl)^–1.min^–1in the sink-limited leaves. These results appear to indicatethat the reduction in photosynthetic activity under sink-limitedconditions was mostly due to a deactivation of RuBPcase. Theactivity of deactivated RuBPcase in the sink-limited leaveswas restored to 4.1 µmole CO₂.(mg Chl)^–1.min^–1by incubation of the enzyme in a medium that contained onlyNa₂HPO₄. This result suggests that free P_i in chloro-plastsplays an important role in the activation of the enzyme. Thelevel of P_i in the sink-limited leaves was 62% of that in thestandard leaves. On the basis of these various results, it appearsthat the deactivation of RuBPcase in the sink-limited leavesis the result of a decrease in the level of P_i. The role offree P_i in the activation of RuBPcase, in particular at atmosphericconcentrations of CO₂, was also investigated. (Received November 30, 1989; Accepted May 11, 1990)     

The Photosynthetic Net Carbon Dioxide Exchange Potential in Conventional and 'Leafless' Phenotypes of Pisum sativum L. in Relation to Foliage Area, Dry Matter Production and Seed Yield

The effect of the ‘leafless’ mutation (in whichtendrils replace leaflets and the stipules are reduced to avestigial form) upon foliage area, photosynthetic net CO₂ uptakepotential, dry matter production and seed yield in Pisum sativumwas studied by comparing two near-isogenic lines of genotypeafafstst and ++++. The mutation is of potential agronomic valuein that it offers improved lodging resistance, crop drying andharvester throughput. In the conventional phenotype the total foliage area of themain axis attained a plateau (456 cm²) at day 56 from seedlingemergence, whereas corresponding values for the ‘leafless’mutant showed a total area of 208 cm² at day 68 with no indicationof a plateau. The agronomic consequence of this is discussed.During the vegetative phase of the plant the maximum CO₂ uptakepotential in the fully expanded conventional leaf was 8·5mg CO₂ leaf^–1 h^–1 and in the ‘leafless’mutant this value was 7·0 mg CO₂ leaf^–1 h^–1.For most ‘leaves’ of the latter phenotype this valuewas between 30 and 60 per cent less than for their conventionalcounterpart. There was a consistently higher photosyntheticpotential per unit area in tendrils of the ‘leafless’mutant than in leaflets of the conventional phenotype. The respectivemean specific values for the two phenotypes were 53 and 37 mgCO₂ dm^–2 h^–1. The problem of obtaining a meaningfulsurface area value for tendrils is discussed and the cylindricalnature of tendrils is taken into account. The ‘leafless’ mutant consistently accumulated 50per cent less dry matter than did conventional plants in theperiod from seedling emergence to anthesis and yield of maturedry seed per plant showed a reduction of 50 per cent both inseed number and total seed weight. The implications for future breeding and selection programmesaimed at haulm reduction are discussed in relation to evaluatingthe ability of the background genotype to produce adequate tendrilsin the presence of afafstst. Triticum aestivum, wheat, callus culture, organogenesis     

Ca2+ sensitivity of BK channels in GH3 cells involves cytosolic phospholipase A2

To test thehypothesis that intracellular Ca²⁺activation of large-conductanceCa²⁺-activatedK⁺ (BK) channels involves thecytosolic form of phospholipase A₂ (cPLA₂), we first inhibited theexpression of cPLA₂ by treating GH₃ cells with antisenseoligonucleotides directed at the two possible translation start siteson cPLA₂. Western blot analysis and a biochemical assay of cPLA₂activity showed marked inhibition of the expression ofcPLA₂ in antisense-treated cells.We then examined the effects of intracellularCa²⁺ concentration([Ca²⁺]_i)on single BK channels from these cells. Open channel probability (P_o) for thecells exposed to cPLA₂ antisenseoligonucleotides in 0.1 µM intracellularCa²⁺ was significantly lower thanin untreated or sense oligonucleotide-treated cells, but the voltagesensitivity did not change (measured as the slope of theP_o-voltagerelationship). In fact, a 1,000-fold increase in[Ca²⁺]_ifrom 0.1 to 100 µM did not significantly increaseP_oin these cells, whereas BK channels from cells in the other treatmentgroups showed a normalP_o-[Ca²⁺]_iresponse. Finally, we examined the effect of exogenous arachidonic acidon theP_oof BK channels from antisense-treated cells. Although arachidonic aciddid significantly increaseP_o,it did so without restoring the[Ca²⁺]_isensitivity observed in untreated cells. We conclude that although [Ca²⁺]_idoes impart some basal activity to BK channels inGH₃ cells, the steepP_o-[Ca²⁺]_irelationship that is characteristic of these channels involves cPLA₂.

     

Effects of Photoperiod and Maturity Genes on Plant Growth, Partitioning, Radiation Use Efficiency, and Yield in Soyabean [Glycine max(L.) Merrill] 'Clark'

Plants of four isolines of soyabean [Glycine max(L.) Merrill]‘Clark’, viz‘L71-920’ (maturity genecomplemente₁e₂e₃ ), ‘L80-5914’ (E₁e₂e₃), ‘Clark’(e₁E₂E₃), and ‘L65-3366’ (E₁E₂E₃), were grown inshort (12.25 h d ^{- 1}natural light) and long days (12.25 h d^{- 1}natural light supplemented with 2.75 h d ^{- 1}low-irradianceartificial light) from first flowering to maturity in a polythenetunnel maintained at 30/24°C (day/night). Whereas therewere few differences among the isolines grown in short days,in long days the dominant alleles increased crop duration, biomassand seed yield substantially. Increases in biological and economicyield were not solely a consequence of longer crop duration:the dominant alleles also increased crop growth rate and radiationuse efficiency in long days (from 1.3 g MJ ^{- 1}total radiationine₁e₂e₃ to 2.8 g MJ ^{- 1}inE₁E₂E₃ ). Greater radiation use efficiencyresulted from a relatively longer leaf area duration, betterdistribution and orientation of a larger mass of leaves withinthe canopy, and smaller partitioning of assimilates to reproductivestructures. The work reveals the substantial effects of thethree lociE₁ / e₁, E₂/ e₂and E₃/e₃ on the response of plantgrowth, as well as development, to environment. Their relevanceto crop adaptation is discussed. Copyright 2000 Annals of BotanyCompany Glycine max(L.) Merrill, soyabean, maturity genes, flowering, phenology, growth, yield