The Significance of Transpirationally Derived Nitrogen in Protein Synthesis in Fruiting Plants of Pea (Pisum sativum L.)

Feeding of ¹⁵N-nitrate, ¹⁵N(amide)-L-glutamine, or ¹⁵N-L-glutamicacid to detached shoots of pea through the transpiration streamresults in the soluble and insoluble nitrogen of stem, leaves,and fruits becoming extensively enriched with isotopic nitrogen.The time course of labelling suggests that non-reproductiveparts are the principal centres of uptake and assimilation andthat from them translocation takes place to the developing seeds. Distribution patterns for ¹⁵N in free and protein-bound aminoacids of leaf and seed indicate that each labelled source donatesnitrogen to a wide range of amino compounds, with no evidenceof consistent differences in the manner in which each is assimilated.Alanine, glutamic acid, homoserine, and -aminobutyric acid,are the main recipients of ¹⁵N in the soluble fraction of theleaves, whilst in the insoluble fraction nitrogen of the aminoacids serine, glycine, alanine, threonine, glutamic acid + glutamine,and aspartic acid + asparagine achieves high specific labelling.Amino acids of the seeds are labelled more uniformly with ¹⁵N. A complementary ¹⁴C-labelling experiment on the translocationof photosynthetically fixed carbon from leaf to seed is describedand the labelling patterns obtained for amino acids in leaf,seed, and phloem exudate are discussed in relation to thosefor ¹⁵N.     

Light-induced Activation of Electrogenic H+ Extrusion and K+ Uptake in Elodea densa Depends on Photosynthesis and is Mediated by the Plasma membrane H+ ATPase

In Elodea densa leaves light strongly stimulates electrogenic,K ⁺-dependent, vanadate- and erythrosin B-sensitive H⁺ extrusionand hyperpolarizes the transmembrane electrical potential. Theseeffects of light are suppressed by treatment with DCMU, an inhibitorof photosynthesis, which has no effect on H⁺ extrusion in thedark. Light-induced H⁺ extrusion requires the presence of K⁺in the medium and is associated with increased K⁺ uptake andalkalinization of the cell sap. Light-induced H⁺ extrusion increaseswith increased CO₂ concentration. At constant CO₂ concentration(10⁴ parts 10^–6) the rate of H⁺ extrusion is stronglyenhanced by an increased light intensity up to 30 W m^–2.Different wavelengths, between 400 and 730 nm, induce a significantstimulation of both proton secretion and transmembrane potentialhyperpolarization. The stimulating effects of light on H⁺ extrusion, K⁺ uptakeand cell sap pH are very similar to those induced in the darkby fusicoccin, a toxin known to stimulate strongly ATP-driven,vanadate- and erythrosin B-sensitive H⁺ transport. In the light,the effects of fusicoccin are only partially additive to thoseof light, thus suggesting that the two factors influence thesame system. The identification of this system with the plasmamembrane H⁺-ATPase is indicated by the observed inhibition ofthe effects of either light or fusicoccin by the H⁺-ATPase inhibitorsvanadate and erythrosin B. These data indicate that the activation of electrogenic H⁺ extrusionand of K⁺ uptake by light is mediated by some products of photosynthesis.The mechanism and the possible physiological implications ofthis phenomenon are discussed. Key words: Photosynthesis, H⁺ pump, K⁺ uptake, Elodea densa     

Some Physiological Aspects of Copper and Zinc Tolerance in Agrostis tenuis Sibth.: Cell Elongation and Membrane Damage

Three clones of Agrostis tenuis Sibth. were studied with respectto the effects of Zn and Cu on the growth of root segments excisedfrom the zone of cell elongation. Elongation growth in segmentsfrom a Cu-tolerant and a Zn-tolerant clone was inhibited toa lesser extent by Cu and Zn respectively than was the growthof a clone which was not tolerant to these metals. Concentrationsof Cu²⁺ which inhibited root growth also caused leakage of K⁺from the cells but toxic concentrations of Zn²⁺ did not induceK⁺ leakage. Copper induced a higher rate of K⁺ leakage at 25than at 0 °C. The impllcations of these results for thesite of the toxic effects of Zn and Cu and the nature of theresistance mechanisms are discussed.     

Induction of Solute Release from Nicotiana tabacum Tissue 10

For determination of the effects of polymyxin B, polymyxin E,or ethylenediamine tetra-acetic acid (EDTA) on plant cell membranes,the rates at which three solutes, K⁺, P₁, and sugar, leakedfrom treated tissue culture cell suspensions of Nicotiana tabacumwere measured. The kinetics of leakage from cells treated witheither of the polymyxins was biphasic, whereas kinetics forcells treated with EDTA was monophasic. Only K⁺ leaked frompolymyxin-treated cells during the first phase, and all threesolutes leaked during the second phase. The slower first phaseis interpreted as leakage of K⁺ from the Donnan free space andcytoplasm, and the faster second phase as the leakage of solutesfrom the vacuole. The monophasic kinetics of EDTA treatmentindicated that solutes were leaking simultaneously from cytoplasmand vacuole. Of the divalent cations tested, only Ca⁺⁺ and Mn⁺⁺counteracted the effects of polymyxin and EDTA. Ca⁺⁺ even restoredP¹ and sugar uptake. Addition of Mg⁺⁺ or Sr⁺⁺ to polymyxin-treatedcells did not stop solute leakage but actually enhanced theleakage rates. A model is presented that suggests that polymyxinor EDTA induces solute leakage by forming pores in plant cellmembranes. The effects of divalent cations on membranes oncethe pores are formed are also discussed. Key words: Polymyxin, EDTA, Nicotiana tabacum, Solute leakage     

Investigations of Carbon Transport in Plants: I. THE USE OF CARBON-11 TO ESTIMATE VARIOUS PARAMETERS OF THE TRANSLOCATION PROCESS

A technique is described which enables several components ofthe translocation system to be measured routinely, in vivo andsimultaneously. Plants are exposed to a 1-min pulse of ¹¹CO₂and the movement of the ¹¹C pulse through the plant followedusing an array of scintillation counters. It is possible toestimate the time taken for the ¹¹C to enter the sieve tubes.In addition, the speed of translocation can be obtained frommeasurements of the arrival of the ¹¹C pulse at different positionsalong the translocation pathway. The increasing half-width ofthe pulse as it enters and moves through the sieve tubes givessome indication of the extent of the delays in the differentparts of the translocation system. The short half-life of the¹¹C allows the measurements to be repeated on the same plantseveral times per day. A technique is discussed whereby dataon the movement of ¹¹C could be combined with simultaneous measurementsof the rate of uptake of carbon dioxide to provide an estimateof the rate of movement of carbon.     

Intracellular Mobilization of Ca2+ and Inhibition of Cytoplasmic Streaming Induced by Transcellular Osmosis in Internodal Cells of Nitella flexilis

When transcellular osmosis was induced in internodal cells ofNitella flexilis that had been rendered inexcitable by treatmentwith KCl or EGTA, the rate of cytoplasmic streaming was reducedand the membrane was depolarized. In both KCl- and EGTA-treatedcells, the endoosmosis induced a significant increase in theconcentration of Ca²⁺ in the cytoplasm, which was demonstratedby monitoring the emission of light from aequorin that had beeninjected into the cytoplasm. When transcellular osmosis was induced in tonoplast-free cells,in which the intracellular Ca²⁺ concentration had been stabilizedat a very low level by treatment with the Ca²⁺-chelating agentEGTA, no change in the rate of cytoplasmic streaming on theendoosmosis side was observed. Hydration of the cytoplasm in the absence of endoosmosis wasinduced by direct introduction of a hypotonic medium into thevacuole by intracellular perfusion. The results mimicked theinhibition of streaming induced by transcellular osmosis. During transcellular osmosis, chloroplasts on the endoosmosisside swelled as a result of dilution of the cell sap. Swellingof chloroplasts was demonstrated to be unrelated to the inhibitionof streaming, since streaming was retarded at sites from whichchloroplasts had been removed. It is suggested that hydration of the cytoplasm triggers themobilization of Ca²⁺ from internal stores and causes an increasein the level of cytoplasmic Ca²⁺ that is responsible for theinhibition of streaming. Possible mechanisms for the osmosis-inducedincreases in the level of Ca²⁺ in the cytoplasm are discussed. (Received January 11, 1993; Accepted November 8, 1993)     

Apical Dominance in Vicia faba

Apical dominance phenomena have been studied in seedlings ofVicia faba particularly in relation to the movement about theplant of uracil-2-¹⁴C applied to the cotyledons. Decapitationjust below the second node releases the growth of the lowermostlateral bud and inhibition is completely reimposed by applicationof indole-3-acetic acid (IAA) to the cut surface. Uracil-2-¹⁴Capplied in solution to the cotyledons is distributed in thestems of all experimental seedlings with no consistent differencesdue to decapitation or IAA application. On the other hand, decapitationresults in a rapid increase in uracil-2-¹⁴C content in the lateralbuds which far exceeds their promoted growth. This uptake iscompletely suppressed by IAA application. A ring of tri-iodobenzoicacid (TIBA)-lanolin paste around the stem above the bud suppressesIAA action both in bud growth and on uracil-2-¹⁴C uptake, andalso on the movement of IAA-1-¹⁴C down the stem. TIBA-application to the base of the bud does not prevent IAAaction on bud growth, but also does not prevent the movementof IAA-1-¹⁴C (or a water soluble product of its metabolism)into the bud. Direct application of kinetin to the lateral bud of intact plantscauses a short-lived release of growth. Gibberellic acid producesa smaller and scarcely significant increase which is additiveto the kinetin effect. Neither has any effect on uracil-2-¹⁴Cmovement into the bud. The implications of these findings are discussed in relationto various existing theories of the mode of auxin action inapical dominance and it is concluded that their strongest supportis for a mechanism involving the suppression of phloem differentiationin the vascular supply to the bud.     

Computer-assisted Video Image Analysis of Spatial Variations in Membrane-associated Ca2+ and Calmodulin during Pollen Hydration, Germination and Tip Growth in Nicotiana tabacum L.

Video images of the distributional pattern of membrane-associatedcalcium (Ca²⁺) and calmodulin (CaM) have been documented andanalysed during pollen hydration, germination and tip growthin Nicotiana tabacum. Digitization of fluorescence microscopeimages of chlorotetracycline (CTC) and fluphenazine (FPZ)-fluorescenceemissions reveal that there is a maximum concentration of membrane-associatedCa²⁺ and also CaM in the vicinity of germination apertures ofhydrated pollen. With the onset of germination relatively higheramounts of Ca²⁺ and CaM were found to regionalize towards theaperture through which the pollen tube would emerge Both shortand long growing pollen tubes manifest tip-to-base Ca²⁺ andCaM gradients which are disturbed in non-growing tubes. Tubegrowth and the Ca²⁺-gradient were significantly affected byvanadate and verapamil suggesting that both a vanadate-sensitiveCa²⁺-transport system and verapamil-sensitive Ca²⁺ channelsare involved in maintaining Ca²⁺ homeostasis during pollen germinationand tube growth. The possible interactions of Ca²⁺ and CaM withdifferent cytoskeletal proteins modulating organelle movementare also briefly discussed. Image analysis, calcium, calmodulin, Nicotiana tabacum L., pollen germination, pollen tube, tip growth, Ca²⁺-channels, Ca²⁺ transport ATPase     

Chloride Transport in Chara corallina and the Electrochemical Potential Difference for Hydrogen Ions

The pH of the cytoplasm of Chara corallina cells has been measuredwith the weak acid 5,5-dimethyloxazolidine-2,4-dione (DM0).Over an external pH range 4·5–9·5 the resultsfit the regression equation pH_cytoplasm=6·28+0·22pH_out. Using measured values of the electric potential difference acrossthe plasmalemma we have calculated the electrochemical potentialdifference across this membrane for H⁺ and Cl^–. Thesedata are used to test the hypothesis that the inward transportof Cl^– is coupled to the inthix of H⁺ or, which comesto the same thing, efflux of OH^–. One-for-one couplingwill not give net Cl^– uptake from solutions with pH greaterthan about 7·2, unless the cytoplasmic Cl^– concentrationis lower than 10 mM, or the pH just outside the membrane islower than that in the bulk solution. It is shown that net Cl^–uptake proceeds from solutions with pH up to 9. The alternative possibility is that Cl^– transport is broughtabout by co-transport of two H⁺ for each Cl^–; this isnot ruled out by the results reported. Such a mechanism mightbe detectable by its electrogenic effect: although such effectshave not been detected, it is shown that they would be smallunder most conditions. Other possible mechanisms are discussed.     

Mitochondrial reactive oxygen species and Ca2+ signaling

Mitochondria are an important source of reactive oxygen species (ROS) formed as a side product of oxidative phosphorylation. The main sites of oxidant production are complex I and complex III, where electrons flowing from reduced substrates are occasionally transferred to oxygen to form superoxide anion and derived products. These highly reactive compounds have a well-known role in pathological states and in some cellular responses. However, although their link with Ca²⁺ is well studied in cell death, it has been hardly investigated in normal cytosolic calcium concentration ([Ca²⁺]_i) signals. Several Ca²⁺ transport systems are modulated by oxidation. Oxidation increases the activity of inositol 1,4,5-trisphosphate and ryanodine receptors, the main channels releasing Ca²⁺ from intracellular stores in response to cellular stimulation. On the other hand, mitochondria are known to control [Ca²⁺]_i signals by Ca²⁺ uptake and release during cytosolic calcium mobilization, specially in mitochondria situated close to Ca²⁺ release channels. Mitochondrial inhibitors modify calcium signals in numerous cell types, including oscillations evoked by physiological stimulus. Although these inhibitors reduce mitochondrial Ca²⁺ uptake, they also impair ROS production in several systems. In keeping with this effect, recent reports show that antioxidants or oxidant scavengers also inhibit physiological calcium signals. Furthermore, there is evidence that mitochondria generate ROS in response to cell stimulation, an effect suppressed by mitochondrial inhibitors that simultaneously block [Ca²⁺]_i signals. Together, the data reviewed here indicate that Ca²⁺-mobilizing stimulus generates mitochondrial ROS, which, in turn, facilitate [Ca²⁺]_i signals, a new aspect in the biology of mitochondria. Finally, the potential implications for biological modeling are discussed. mitochondria; calcium     

Photosynthetic Fixation of 14Carbon by Internodal Cells of Chara corallina

Maximum fixation rates of 120 and 60 pmol cm^–2 s ^–1wereobtained when exogenous carbon was supplied as ¹CO₂ and H¹⁴CO₃^–respectively. These values are considerably higher than thosepreviously reported for this species. A kinetic analysis wasperformed on this data. Substrate saturation in the concentrationrange 1.0–1.5 mM was observed for both CO₂ and HCO₃^– In the presence of exogenous CO², a linear relationship wasobserved between light intensity and fixation while the HCO₃^–relationship was slightly sigmoidal. Fixation saturated at intensitiesof 15–20 W m^–2 and 13–15 W m^–2 for exogenous¹⁴CO² and H¹⁴CO₃^–respectively. The presence, in this species, of an extremely active HCO₃^–transport system, situated in the plasmalemma, demonstratesthat when alkaline solutions are employed the involvement ofthis ion cannot be ignored during electrical studies on thismembrane. The maximum H¹⁴CO₃^– influxes obtained duringthis study are the largest ionic fluxes measured for any Characeanspecies. It was demonstrated that CO² for fixation can be supplied simultaneouslyby gaseous diffusion and HCO₃^– transport (cf. Raven, 1968).Inhibition of H¹⁴CO₃^– influx was observed in the presenceof Tris, Tricine, and borate buffers, and CO₃^{2 –} alsoappeared to act as a strong inhibitor. The possible mechanism(s)by which this inhibition occurs is discussed.     

Patch-Clamp Study on a Ca2+-Regulated K+ Channel in the Tonoplast of the Brackish Characeae Lamprothamnium succinctum

Cytoplasmic drops were prepared from internodal cells of thebrackish Characeae Lamprothamnium succinctum. Applying the patch-clamptechnique to single drops covered with tonoplast, we demonstratedthe presence of Ca²⁺-regulated K⁺ channels in the tonoplast.In a cell-attached mode, the selectivity of such channels forK⁺ was about 50 times that for Na⁺. This channel showed a tendencyto rectify in an outward direction. In the negative region ofthe pipette voltage, the conductance of this channel was 50pS, while it was 100 pS in the positive voltage region. Whenthe pipette voltage was increased above 50 mV, two conductancelevels were found in the cell-attached mode as well as in theexcised patch (cytoplasmic-side-out patch), which was obtainedby pulling the patch pipette from the cytoplasmic drop underconditions of low levels of Ca²⁺. Using the excised patch, wecontrolled the level of Ca²⁺ on the cytoplasmic side of thechannels. At a low level of Ca²⁺ (pCa=8) on the cytoplasmicside, the open frequency was very low and the opening time wasshort. An increase in Ca²⁺ on the cytoplasmic side (pCa = 5)increased both the frequency and the duration of opening. However,the conductance of the channels did not change. This regulationby Ca²⁺ of the K⁺ channels was reversible, that is, additionof EGTA on the cytoplasmic side inactivated the channels. Thepresent study demonstrates a direct action of Ca²⁺ on the K⁺channels. The physiological role of the K⁺ channel in the regulationof turgor in Lamprothamnium is discussed. (Received January 9, 1989; Accepted March 8, 1989)     

Differential effect of Calcium and Magnesium on Mechanical Properties of Pea Stem Cell Walls

Replacement of calcium ion with magnesium ion in the cell wallof the pea epicotyl makes the wall more extensible. A possiblerole of this differential effect of Ca²⁺ and Mg^2– in regulatingcell elongation in pea epicotyl is discussed. The ratio of the content of calcium ion to that of magnesiumion (Ca²⁺/Mg²⁺) in the walls decreased markedly in the orderof the first > the second > the third internodes of thepea epicotyl. The capacity of the walls for cation exchangeincreased in the same order, whereas the calcium-magnesium ionexchange selectivity of the walls was virtually constant. Ourresults indicate that the changes in the Ca²⁺/Mg²⁺ ratio amongthe internodes is not attributable to the ion exchange propertiesof the walk per se, but is due to other physiological conditionswhich regulate the activities of free calcium and magnesiumions in the environment with which the walls are in equilibrium. ¹ Present address: Wakayama Research Laboratories, Kao SoapCo., Ltd., Wakayama 640-91, Japan ² Present address: Foold Development Laboratories, Meiji SeikaKaisha Ltd., Kawasaki 210, Japan (Received May 1, 1981; Accepted August 26, 1981)     

Malate: A Possible Source of Error in the NAD Glutamate Dehydrogenase Assay

The effects of externally induced metabolic perturbations areoften studied through changes of the enzyme activity patternsin crude plant extracts. From glutamate dehydrogenase (GDH)it is reported that environmental changes not only influencethe amount of the enzymatic activity, but also the ratio ofthe aminating to the deaminating activities (NADH/NAD⁺ ratio).Using crude cell extracts of suspension cultures of wheat (Triticumaestivum L. cv. Heines Koga II) we find evidence that the pretreatmentof the homogenate directly influences this ratio. Dialysis ofthese crude cell extracts resulted in a 70% loss of the NAD⁺activity, while the NADH activity remained unchanged. The deaminatingactivity in the dialysed extract could be completely restoredupon addition of a dialysable factor which was identified tobe malate. The interference of malate with the glutamate dehydrogenasereaction is caused through the action of malate dehydrogenaseand glutamate oxaloacetate transaminase which are both presentin high activities in the extracts. Only in exhaustively dialysedcell extracts can the proper deaminating GDH activity be determined.The results are discussed in the light of the controversialreports on the variable ratio of the NADH/NAD⁺ activity of GDH. Key words: Glutamate dehydrogenase, malate, NADH/NAD⁺, activity, Triticum aestivum     

Competitive inhibition of the nickel-induced response to choline by calcium ions in single water fibers of the frog glossopharyngeal nerve

NiCl₂ induces a response to cboline Cl and enhances the responseto CaCl₂ in water-sensitiv fibers (water fibers) of the frogglossopharyngeal nerve. The Ni²⁺-induced choline⁺ response wasinhibited by Ca²⁺ ions and, conversely, the enhanced Ca²⁺ responseby Ni²⁺ ions was inhibited by choline⁺ ions. Hence, there existsa mutual antagonism between Ca²⁺ and choline⁺ ions. In the presentstudy, the inhibition of the Ni²⁺-induced choline⁺ responseby Ca²⁺ ions was investigated quantitatively. The assumptionwas made that receptors for choline (X_Ch) exist and that bindingof a choline⁺ ion to X_Ch, brings about a neural response. Itwas further assumed that the magnitude of the neural responseis proportional to the amount of choline-X_Ch, complex minussome constant (the threshold concentration of the choline-X_Ch,complex). The results from analysis of double-reciprocal plotwere consistent with the hypothesis that Ca²⁺ ions compete withcholine⁺ ions for X_Ch,. The dissociation constants for the choline-X_Ch,complex and the CaX_Ch, complex were obtained to be 0.6 M and7.4 x 10^-5 M, respectively. This result indicates that the affinitiesof X_Ch, for choline⁺ and Ca²⁺ ions are very different. Furthermore,Mg²⁺ ions did not affect the Ni²⁺-induced choline⁺ response,an indication that the affinity of X_Ch, is not charge-specific,but is chemically specific. The identification of a competitiveinhibitor of the choline⁺ response provide* evidence for existenceof a choline-specific receptor at the surface of taste cellsthat are innervated by the water fibers of the frog glossopharyngealnerve. Differences between the features of the response to cholineCl in the chorda tympani nerve of the rat and those in the frogglossopharyngeal nerve are discussed.     

Efficiency of K+ Utilization by Barley Varieties: Activation of Pyruvate Kinase

Barley (Hordeum vulgare L.) varieties differed in their raponseto [K⁺]₀, in terms of their utilization efficiencies (UE = freshweight. concentration of [K⁺]₁^–1). At low [K⁺]₀, Compana,an efficient-non-responder demonstrated superior utilizationof absorbed K⁺. On the other hand, at high [K⁺]₀, Fergus (anefficient responder) and BT 334 (an inefficient responder) hadhigher UE values for K⁺ than Compana which performed poorlyat this [K⁺]₀. Kinetic parameters for K⁺ activation of the enzyme pyruvatekinase from 12 barley varieties, representing a range of UEvalues, were determined. Varieties showed substantial differencesin their V_max values (P<0·01). Compana, an efficientvariety, had the highest V_max (31 µmol g^–1 freshwt. h^–1) which was about 50% higher than that of Mingo,an inefficient variety. By contrast, K_m values for the enzymeswere not significantly different among varieties The mean valuesfor all varieties (3·9±0·15 mol m^–3K⁺) is far below the estimated cytoplasmic [K⁺] (100-200 molm^–3). It is, therefore, unlikely that differences in theutilization of K⁺ by these varieties can be explained on thebasis of differential requirements for (K⁺) activation of theseenzymes. Alternative possibilities for differences in the utilizationof K⁺ are discussed. Key words: K⁺ utilization efficiency, Pyruvate kinase, Barley varieties     

Wavelength effects on photosynthetic carbon metabolism in Chlorella

To study the wavelength-effect on photosynthetic carbon metabolism,¹⁴C-bicarbon-ate was added to Chlorella vulgaris 1 lh suspensionunder monochromatic blue (456 nm) and red (660 nm) light. Thelight intensities were so adjusted that the rates of ¹⁴CO₂ fixationunder blue and red light were practically equal. Analysis of¹⁴C-fixation products revealed that the rates of ¹⁴CO₂ incorporationinto sucrose and starch were greater under red light than underblue light, while blue light specifically enhanced ¹⁴CO₂ incorporationinto alanine, aspartate, glutamate, glutamine, malate, citrate,lipid fraction and alcohol-water insoluble non-carbohydratefraction. Pretreatment of the algal cells in phosphate mediumin the dark, which was essential for the blue light enhancementof PEP carboxylase activity, was not necessary to induce theabove wavelength effects. Superimposition of monochromatic bluelight at low intensity (450 erg.cm^–2.sec^–1) on thered light at saturating intensity caused a significant decreasein the rate of ¹⁴CO₂ incorporation into sucrose and increasein incorporation into alanine, lipid-fraction, aspartate andother related compounds, indicating that the path of carbonin photosynthesis is regulated by short wavelengdi light ofvery low intensity. Possible effects of wavelength regulationof photosynthetic carbon metabolism in algal cells are discussed. ¹ Part of this investigation was reported at the XII InternationalBotanical Congress, Leningrad, 1975 and the Japan-US CooperativeScience Seminar "Biological Solar Energy Conversion", Miami,1976. Requests for reprints should be addressed to S. Miyachi,Radioisotope Centre, University of Tokyo, Bunkyo-ku, Tokyo 113,Japan. ⁴ Present address: Department of Chemistry, Faculty of PharmaceuticalSciences, Teikyo Univ., Sagamiko, Kanagawa, Japan. (Received August 6, 1977; )     

The rice tapetum-specific gene RA39 encodes a type I ribosome-inactivating protein

A tapetum-specific cDNA encoded by a rice gene, RA39, was isolated by cDNA subtractive hybridization, differential screening and rapid amplification of cDNA ends. RA39 is a single-copy gene in the rice genome. mRNA in situ hybridization indicates that this gene is a tapetum-specific gene, and highly expressed in the tapetal cells at the meiosis and tetrad stages. The RA39 cDNA is 1,013 bp in length with an open reading frame encoding 298 amino acid residues. This cDNA sequence does not show significant homology to any known sequences in GenBank databases, but its deduced amino acid sequence (RA39) has between 19 and 34% sequence identity to ribosome-inactivating proteins (RIPs). Optimal alignment reveals that the five amino acid residues constituting the active site of the ricin A-chain (Tyr⁸⁰, Tyr¹²³, Glu¹⁷⁷, Arg¹⁸⁰ and Trp²¹¹), which are invariant among all RIPs published to date, are conserved in RA39. Recombinant RA39 protein expressed in Escherichia coli was purified to homogeneity. The purified protein exhibits the RNA N-glycosidase activity of RIPs. This demonstrates that RIPs occur in the reproductive organs of rice. The possible function of RA39 in anther development is discussed.     

Characterization of ATPase Activity Associated with Plasma Membrane from Vigna Hypocotyls

A plasma membrane fraction was isolated from the hypocotylsof cowpea {Vigna unguiculata) by a combination of differentialcentrifugation and sucrose density gradient centrifugation.The ATPase activity of this fraction was dependent on divalentcations (Mn²⁺>Mg²⁺>Co²⁺>Ca²⁺>Fe²⁺>Zn²⁺>Ni²⁺)but was not further stimulated by monovalent cations (K⁺ and/orNa⁺). The pH optimum for the activation of ATPase by Mg²⁺ was7.0. This fraction hydrolyzed ATP or UTP as a substrate andthe ATPase activity obeyed a Michaelis-Menten type of kinetics.The K_m for MgATP ranged from 0.65 to 1.1 mM. The ATPase activitywas inhibited by inhibitors such as N, N'- dicyclohexylcarbodiimide,diethylstilbestrol and triphenyltin chloride, all of which arereported to block proton (H⁺) transport in plant cells, butwas insensitive to those of mitochondrial ATPase such as oligomycinand sodium azide. The ATPase activity was not stimulated bytreatment with ionophores (e.g., carbonyl cyanide p-trifluoromethoxyphenylhydrazone,3,5-di-ter-butyl-4-hydroxybenzilidenemalononitrile and valinomycin⁺KCl)which would be expected to dissipate the electrochemical potentialdifference of H⁺ or the membrane potential difference. The characteristics of the ATPase are compared with those ofplasma membrane ATPases of other plants and its possible rolein H⁺-transport is discussed. ¹ Present address: Institute of Applied Biochemistry, Yagi MemorialPark, Mitake, Gifu 505-01, Japan or Laboratory for Plant EcologicalStudies, Faculty of Science, Kyoto University, Kyoto 606, Japan. (Received April 20, 1984; Accepted August 14, 1984)     

Ion Imbalance in Capsicum annuum, scabrous diminutive, a Wilty Mutant of Pepper I. SODIUM FLUXES

Leaves of the wilty pepper mutant, scabrous diminutive, accumulatemore Na+ than those of the normal genotype, when both grow inmedium containing N⁺. It seems that the regulation of Na⁺ fluxin the mutant root was modified. Net uptake of Na⁺ was muchhigher and efflux of ²²Na⁺ was lower in the mutant roots thanin the normal ones. Two possible explanations for these differencesbetween mutant and normal plants are discussed, namely (a) achange in membrane permeability and (b) a change in the mechanismof Na⁺ extrusion.