Cellular Growth in Roots of a Gibberellin-Deficient Mutant of Tomato (Lycopersicon esculentum Mill.) and its Wild-type

The role of gibberellins in regulating the growth of tomatoroots was investigated by comparing various cellular parametersin cultured roots of the gibberellin-deficient mutant gib-l/gib-lwith those in roots of the near-isogenic wild-type. In addition,wild-type roots treated with 0?1 µM 2S,3S paclobutrazol,an inhibitor of gibberellin biosynthesis, and mutant roots treatedwith 0?1 µM GA₃ were also compared: the former roots constitutea phenocopy of the mutant, whereas the latter roots appear tobe ‘normalized’ and similar to wild-type. The elongationof mutant and phenocopied roots were similar, their maximumelongation rates being about half or two-thirds that of wild-typeor GA₃-treated mutant roots, respectively. These rates wereinterpreted in terms of the numbers and lengths of cells withinthe meristematic and non-meristematic portions of the elongationzone. Mean meristem length tended to be shorter in both themutant and the 2S,3S paclobutrazol-treated wild-type roots thanin the other two types of root. A major difference between thetwo pairs of mutant and normal roots was their mean final celllengths: mean lengths of cortical cells of the mutant and 2S,3Spaclobutrazol-treated roots were, respectively, 39% and 25%shorter than the mean length of wild-type roots. Final celllength in the GA³-treated mutant roots were similar to wild-type.By contrast, the diameters of mature cortical cells of the mutantand phenocopy were about 20% greater than the diameters of equivalentwild-type or ‘normalized’ mutant cells. The meanvolumes of cortical cells in all four types of roots showedno significant differences. Knowledge of the distribution ofcortical cell lengths, widths and volumes along the root axis,together with information about the rate of root elongation,permitted comparisons of the relative elemental growth ratesof each of these three cellular parameters. The available evidence suggests that the level of endogenousgibberellins in mutant roots is lower than in wild-type roots.The present results, therefore, suggest that endogenous gibberellinsare necessary for normal growth of cultured tomato roots andthat they regulate the relative amounts of growth at the longitudinaland transverse walls of the cells which, in turn, affects theshape of the elongating cells. Key words: Cell growth, cultured roots, gibberellin, gib-l mutant, Lycopersicon esculentum, 2S,3S paclobutrazol, relative elemental growth rate, root meristem     

Structural and Kinetic Properties of NADP-Malic Enzyme from the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum L.

NADP-malic enzyme (EC 1.1.1.40 [EC] ), which is involved in Crassulaceanacid metabolism (CAM), was purified to electrophoretic homogeneityfrom the leaves of the inducible CAM plant Mesembryanthemumcrystallinum. The NADP-malic enzyme, which was purified 1,146-fold,has a specific activity of 68.8 µmol (mg protein)^–1min^–1. The molecular weight of the subunits of the enzymewas 64 kDa. The native molecular weight of the enzyme was determinedby gel-filtration to be 390 kDa, indicating that the purifiedNADP-malic enzyme is a hexamer of identical subunits. The optimalpH for activity of the enzyme was around 7.2. Double-reciprocalplots of the enzymatic activity as a function of the concentrationof L-malate yielded straight lines both at pH 7.2 and at pH7.8 and did not reveal any evidence for cooperativity of bindingof L-malate. The K_m value for L-malate was 0.35 mM. Hill plotsof the activity as a function of the concentration of NADP⁺indicated positive cooperativity in the binding of NADP⁺ tothe enzyme with a Hill coefficient (nH) of 2.0. An S_0.5 value(the concentration giving half-maximal activity) of 9.9 µMfor NADP⁺ was obtained. Oxaloacetate inhibited the activityof the NADP-malic enzyme. Effects of succinate and NaHCO₃ onthe activity of NADP-malic enzyme were small. (Received October 30, 1991; Accepted May 1, 1992)     

Altered pH(i) regulation and Na(+)/HCO3(-) transporter activity in choroid plexus of cilia-defective Tg737(orpk) mutant mouse

Tg737^orpk mice have defects in cilia assembly and develop hydrocephalus in the perinatal period of life. Hydrocephalus is progressive and is thought to be initiated by abnormal ion and water transport across the choroid plexus epithelium. The pathology is further aggravated by the slow and disorganized beating of motile cilia on ependymal cells that contribute to decreased cerebrospinal fluid movement through the ventricles. Previously, we demonstrated that the hydrocephalus phenotype is associated with a marked increase in intracellular cAMP levels in choroid plexus epithelium, which is known to have regulatory effects on ion and fluid movement in many secretory epithelia. To evaluate whether the hydrocephalus in Tg737^orpk mutants is associated with defects in ion transport, we compared the steady-state pH_i and Na⁺-dependent transport activities of isolated choroid plexus epithelium tissue from Tg737^orpk mutant and wild-type mice. The data indicate that Tg737^orpk mutant choroid plexus epithelium have lower pH_i and higher Na⁺-dependent HCO₃^– transport activity compared with wild-type choroid plexus epithelium. In addition, wild-type choroid plexus epithelium could be converted to a mutant phenotype with regard to the activity of Na⁺-dependent HCO₃^– transport by addition of dibutyryl-cAMP and mutant choroid plexus epithelium toward the wild-type phenotype by inhibiting PKA activity with H-89. Together, these data suggest that cilia have an important role in regulating normal physiology of choroid plexus epithelium and that ciliary dysfunction in Tg737^orpk mutants disrupts a signaling pathway leading to elevated intracellular cAMP levels and aberrant regulation of pH_i and ion transport activity. cAMP; ion transport     

Protein kinase D inhibits plasma membrane Na+/H+ exchanger activity

The regulation of plasma membraneNa⁺/H⁺exchanger (NHE) activity by protein kinase D (PKD), a novel proteinkinase C- and phorbol ester-regulated kinase, was investigated. Todetermine the effect of PKD on NHE activity in vivo, intracellular pH(pH_i) measurements were made inCOS-7 cells by microepifluorescence using the pH indicator cSNARF-1.Cells were transfected with empty vector (control), wild-type PKD, orits kinase-deficient mutant PKD-K618M, together with green fluorescentprotein (GFP). NHE activity, as reflected by the rate of acid efflux(J_H), wasdetermined in single GFP-positive cells following intracellularacidification. Overexpression of wild-type PKD had no significanteffect on J_H(3.48 ± 0.25 vs. 3.78 ± 0.24 mM/min in control atpH_i 7.0). In contrast,overexpression of PKD-K618M increasedJ_H (5.31 ± 0.57 mM/min at pH_i 7.0;P < 0.05 vs. control). Transfectionwith these constructs produced similar effects also in A-10 cells,indicating that native PKD may have an inhibitory effect on NHE in bothcell types, which is relieved by a dominant-negative action ofPKD-K618M. Exposure of COS-7 cells to phorbol ester significantlyincreased J_H in control cells but failed to do so in cells overexpressing either wild-type PKD (due to inhibition by the overexpressed PKD) or PKD-K618M(because basal J_Hwas already near maximal). A fusion protein containing the cytosolicregulatory domain (amino acids 637-815) of NHE1 (the ubiquitousNHE isoform) was phosphorylated in vitro by wild-type PKD, but with lowstoichiometry. These data suggest that PKD inhibits NHE activity,probably through an indirect mechanism, and represents a novel pathwayin the regulation of the exchanger.

     

Simultaneous CO2- and 16O2/18O2-gas exchange and fluorescence measurements indicate differences in light energy dissipation between the wild type and the phytochrome-deficient aurea mutant of tomato during water stress

The CO₂-, H₂O- and ¹⁶O₂/¹⁸O₂ isotopic-gas exchange and the fluorescencequenching by attached leaves of the wild-type and of the phytochrome-deficienttomato aurea mutant was compared in relation to water stressand the photon fluence rate. The chlorophyll content of aurealeaves was reduced and the ultra-structure of the chloroplastswas altered. Nevertheless, the maximum rate of net CO₂ uptakein air by the yellow-green leaves of the aurea mutant was similarto that by the dark-green wild-type leaves. However, less O₂was produced by the leaves of the aurea mutant than by leavesof the wild-type. This result indicates a reduced rate of photosyntheticelectron flux in aurea mutant leaves. No difference in bothphotochemical and non-photochemical fluorescence quenching wasfound between wild-type and aurea mutant leaves. Water stresswas correlated with a reversible decrease in the rates of bothnet CO₂ uptake and transpiration by wild-type and aurea mutantleaves. The rate of gross ¹⁶O₂ evolution by both wild-type andaurea mutant leaves was fairly unaffected by water stress. Thisresult shows that in both wild-type and aurea leaves, the photochemicalprocesses are highly resistant to water stress. The rate ofgross ¹⁸O₂ uptake by wild-type leaves increased during waterstress when the photon fluence rate was high. Under the sameconditions, the rate of gross ¹⁸O₂ uptake by ^aurea mutant leavesremained unchanged. The physiological significane of this differencewith respect to the (presumed) importance of oxygen reductionin photoprotection is discussed. Key words: Water stress, gas exchange, fluorescence quenching, Lycopersicon esculentum, mutant (tomato, aurea), energy dissipation     

STUDIES ON HOMOSERINE DEHYDROGENASE IN PEA SEEDLINGS

Partially purified homoserine dehydrogenase was prepared frompea seedlings. The optimum pH for this enzyme is approximately 5.4. The K_mvaluesfor ASA and TPNH are 4.6xl0^–4Af and 7.7xl0^–5M, respectively.This enzyme can also utilize DPNH but less effectively thanTPNH. In contrast with yeast homoserine dehydrogenase whichis insensitive to — SH reagents, the pea enzyme is inhibitedalmost completely by 10^–4MPCMB and 10^–5MHgCl₂, theinhibition being removed by 10^–2M thioglycolate. Homoserinedehydrogenase was found not only in decotylized seedlings, butalso in cotyledons. The significance of this enzyme in homoserine biosynthesis ingerminating pea seeds has been discussed. (Received February 20, 1961; )     

New fava bean guard cell signaling mutant impaired in ABA-induced stomatal closure

We isolated a mutant from Vicia faba L. cv. House Ryousai. Itwilts easily under strong light and high temperature conditions,suggesting that its stomatal movement may be disturbed. We determinedresponses of mutant guard cells to some environmental stimuli.Mutant guard cells demonstrated an impaired ability to respondto ABA in 0.1 mM CaCl₂ and stomata did not close in thepresence of up to 1 mM ABA, whereas wild-type stomata closedwhen exposed to 10 µM ABA. Elevating external Ca²⁺caused a similar degree of stomatal closure in the wild typeand the mutant. A high concentration of CO₂ (700 µlliter^–1) induced stomatal closure in the wild type, butnot in the mutant. On the basis of these results, we proposethe working hypothesis that the mutation occurs in the regiondownstream of CO₂ and ABA sensing and in the region upstreamof Ca²⁺ elevation. The mutant is named fia (fava bean impairedin ABA-induced stomatal closure). ³ Corresponding author: E-mail, smoiwai{at}agri.kagoshima-u.ac.jp;Fax, +81-99-285-8556.     

Reproductive Allometry in Soybean, Maize and Sunflower

We compared the relationship between grain yield per plant (Y_P)and shoot biomass per plant (S_P) in three annual crops withcontrasting reproductive strategies: sunflower, a determinatespecies with a single inflorescence; maize, a determinate specieswith a limited capacity to adjust the number of ears in responseto resource availability; and indeterminate soybean, a specieswith a large capacity to adjust the number of inflorescences.Our working hypotheses were: H₁—the relationship betweenY_PandS_P is linear; H₂—the intercept of the model is zero,i.e. there is not a threshold plant mass for reproduction. Awide range of Y_Pand S_Pwas generated by manipulation of plantdensity;S_Pvaried between 0.3 and 196 g per plant in soybean,between 6 and 873 g per plant in sunflower and between 23 and697 g per plant in maize. Within these broad ranges of plantsize, both hypotheses were rejected in five out of six experiments,i.e. the relationship between Y_Pand S_Pdeparted from linearityand there was a threshold for S_Pbelow which no grain set occurred.TheS_P threshold for grain set varied widely among species; itwas close to 2 g per plant for soybean, 27 g per plant for sunflowerand 43–71 g per plant for maize. Because of this sizethreshold and non-linearity, harvest index (HI = Y_PS_P^-1) wasstable for mid-size plants, diminished slightly for large plants,and diminished sharply for smaller plants in all three crops.Harvest index stability was highest in soybean, intermediatein sunflower and lowest in maize. Differential stability ofreproductive partitioning partially derived from contrastingpatterns of meristem allocation. Copyright 2000 Annals of BotanyCompany Helianthus annuus L., Zea mays L., Glycine max(L.) Merrill, grain yield, harvest index, plant density, reproductive allocation, meristem allocation, plasticity     

Ion and Glycerol Concentrations in 12 Isolates of Dunaliella

Ginzburg, M., and Ginzburg, B. Z., 1985. Ion and glycerol concentrationsin 12 isolates of Dunaliella.—J. exp. Bot. 36: 1064–1074. Twelve isolates of Dunaliella with average cell volumes rangingfrom 50 to 1400x10^–18 m³ were grown in batch culture at0.5 M or 2.0 M NaCl. Glycerol and ions (Na⁺, K⁺, Mg²⁺, CI^–,phosphate) were measured in log-phase cultures. The contentsof Mg²⁺, K⁺ and phosphate per cell were found to be a functionof cell-volume. Cell glycerol, Na⁺ and Cl^– were functionsof cell-volume and of the NaCl concentration in the medium.Solute concentrations were calculated from the measured cell-volumesand from the ³H₂O content of pellets corrected for intercellularspace using Blue Dextran. Cell glycerol was found to accountfor about one-half of the expected osmolarity, the remainderbeing largely accounted for by Na⁺ and CI^–. Key words: —Dunaliella, isolates, glycerol, ion concentrations     

Temperature Response of Early Foliar Expansion of Potato and Wheat

We studied the course of early leaf area expansion and specificleaf area (S_LA) in potato (Solanum tuberosum L.) and wheat (Triticumaestivum L.) genotypes and tested whether air temperature explainsdifferences in these courses within different environments.Such knowledge can be used to improve crop growth modelling.The relative rate of leaf area expansion (R_L) of potato andwheat decreased with thermal time, but was nearly linear upto a leaf area index (L) of 1.0. TheR_L (L < 1; mean: 17.9x 10^-3°C^-1 d^-1) of potato showed an interaction betweengenotype and environment, and varied with year. TheR_L (L <1; mean: 7.1 x 10^-3°C^-1 d^-1) of winter wheat was lower thanthat of spring wheat (mean: 10.9 x 10^-3°C^-1 d^-1), and bothvaried with year. S_LAof potato increased nearly linearly withthermal time from 5 to 15 m² kg^-1at 50% emergence, to 20 to25 m² kg^-1at 155°Cd, and then decreased slightly. The S_LAofboth winter and spring wheat began at 16 to 23 m² kg^-1and inmost cases increased slightly with thermal time. In potato,regression parameters of S_LAwith thermal time were affectedby environment (management conditions and year) and genotype;in wheat they were affected by environment (year and site).Treatment effects on R_Lof potato were not correlated with thoseon S_LA , and were only partly correlated for wheat. Thereforewe conclude that the early foliar expansion of potato is associatedwith a strong increase in S_LA , and not so for wheat. For bothcrops the course of early leaf area expansion and ofS_LA withair temperature is not robust over environments and genotypes.The consequences of these results for modelling are discussed.Copyright 2000 Annals of Botany Company Triticum aestivum, spring wheat, winter wheat, Solanum tuberosum, leaf area expansion, specific leaf area, early growth, genotype, environment, modelling     

Allocation of Organ Biomass in Perfect and Imperfect Flowers

Perianth M_P, gynoecium M_G, and androecium M_A dry-weight biomass(in g) of 39 species of perfect flowers was measured. Thesedata were pooled with published data from an additional 51 speciesand used to determine size-dependent variations in (M_G and M_A)in terms of the hypothesis that the quotient of M_G and M_A exceeds1·0 for out-breeding (xenogamous) species and less than1·0 for in-breeding (autogamous) species. Ordinary leastsquare regression of the pooled data (n = 90) showed M_G = 0·118M^0·916_P (r² = 0·884) and M_A = 0·186 M^0·975_P(r² = 0·865), indicating that the biomass of the gynoeciumproportionally decrease as floral size increases. The exponentsof these regressions indicate that the ratio of gynoecial toandroecial biomass decreased with increasing floral size suchthat comparatively small flowers (M_P < 0·0021 g) hadM_G/M_A > 1·0 (predicted for 'out-breeders') while comparativelylarger flowers (M_P > 0·0021 g) had M_G /M_A < 1·0(predicted for 'in-breeders'). Thus, on average, the type ofbreeding system was a size-dependent phenomenon. To test whether the biomass of a floral organ-type is a legitimateindicator of gender reproductive effort, the biomass (in g)of stamen filaments M_m and anther sacs M_AS of 39 species wasdetermined. Least square regression of these data showed M_AS= 0·188 M^0·854_fil (r² = 0·967), indicatingthat species with larger stamen filaments, on the average, boreproportionally smaller anther sacs and thereby cautioning againstthe uncritical use of the allocation of biomass to floral organ-typeas a strict gauge of gender-function investment. To determine whether the loss of one gender-function resultsin proportional reallocation of biomass to the remaining gender-function,the size-dependency of androecial and gynoecial biomass wasdetermined for a total of 33 perfect and imperfect flowers ofCucumis melo. Regression of the data obtained from perfect flowersyielded M_A = 0·402 M^1·47_P (r² = 0·898)and M_G = 4·63 M^1·36_P (r² = 0·842). SinceM_G/M_A M^0·11_P , the biomass allocation to the gynoeciumrelative to the androecium decreased with increasing floralsize. This result was consistent with the broad interpecificcomparison based on 90 species with perfect flowers . Regressionof the data for imperfect flowers yielded M_A = 0·151M^1·02_P (r² = 0·675) and M_G = 4·68 M^1·47_P(r² = 0·996), indicating a near allometric relation forthe androecium and a strong positive anisometry for the gynoecium.Thus, for flowers of comparable size, a loss of female genderobtains a modest to significant again in androecial biomasswhereas the loss of male gender yields only a slight increasein gynoecial biomass. Collectively, the results of these studies indicate that biomassallocation patterns are size-dependent phenomena whose complexitieshave been largely ignored in the literature.Copyright 1993,1999 Academic Press Allometry, floral biomass, reproduction     

RNA Polymerase Activity During Breakage of Seed Dormancy by Low Temperature Treatment of Fruits of Acer platanoides (Norway Maple)

Slater, R. J. and Bryant, J. A. 1987. RNA polymerase activityduring breakage of seed dormancy by low temperature treatmentof fruits of Acer platanoides (Norway maple).—J. exp.Bot. 38:1026–1032. Endogenous RNA polymerase activity has been characterized innuclei isolated from embryo axes of Acer platanoides. Optimalactivity was recorded at 4·0 mol m^–3 MgCl₂ and50 mol m^–3 (NH₄)₂SO₄ and total activity could be inhibitedby up to 30% by -amanitin. Stratification of fruits leads toa stimulation of RNA polymerase activity. A minimum of 3 d coldtreatment is required with at least 3-fold stimulation recordedafter 10 d at 4°C. The increased enzyme activity is resistantto -amanitin suggesting an effect on RNA polymerase I. Key words: Acer platanoides, RNA polymerase, seed dormancy     

The Permeability of Ammonia, Methylamine and Ethylamine in the Charophyte Chara corallina (C. australis)

Ritchie, R. J. 1987. The permeability of ammonia, methylamineand ethylamine in the charophyte Chara corallina (C. australis).—J.exp. Bot. 38: 67–76 The permeabilities of the amines, ammonia (NH₃), methylamine(CH₃NH₂) and ethylamine (CH₃CH₂NH₂) in the giant-celled charophyteChara corallina (C. australis) R.Br. have been measured andcompared. The permeabilities were corrected for uptake fluxesof the amine cations. Based on net uptake rates, the permeabilityof ammonia was 6?4?0?93 µm s^–1 (n = 38). The permeabilitiesof methylamine and ethylamine were measured in net and exchangeflux experiments. The permeabilities of methylamine were notsignificantly different in net and exchange experiments, norto that of ammonia (P_methylamine = 6?0?0?49 µm s^–1(n = 44)). In net flux experiments the apparent permeabilityof ethylamine was slightly greater than that of ammonia andmethylamine (P_{ethylamine, net} = 8?4?1?2 µm s^–1 (n= 40)) but the permeability of ethylamine based on exchangeflux data was significantly higher (P_{ethylamine, exchange} =14?1?2 µm s^–1 (n = 20)). Methylamine can be validlyused as an ammonium analogue in permeability studies in Chara. The plasmalemma of Chara has acid and alkaline bands; littlediffusion of uncharged amines would occur across the acid bands.The actual permeability of amines across the alkaline bandsis probably about twice the values quoted above on a whole cellbasis i.e. the permeability of ammonia across the permeablepart of the plasmalemma is probably about 12 µm s^–1. Key words: Chara, permeability, ammonia, methylamine     

Preliminary characterization of 1-aminocyclopropane-1-carboxylate oxidase properties from embryonic axes of chick-pea (Cicer arietinum L.) seeds

For a deeper understanding of the germination of chick–pea(Cicer arietinum) seeds, which is dependent upon ethylene synthesis,a crude extract containing authentic ACC oxidase (ACCO) activitywas isolated in soluble form from the embryonic axes of seedsgerminated for 24 h. Under our optimal assay conditions (200mM HEPES at pH 7.0, 4µM FeS0₄, 6 mM Na–ascorbate,1 mM ACC, 20% 0₂, 3% CO₂ , and 10%glycerol) this enzyme was5–fold more active than under the conditions we used initiallyin the present work. The enzyme has the following K_m: 28 µMfor ACC (approximately 4–fold less than in vivo), 1.2%for O₂ (in the presence of an optimal CO₂ concentration of 3%),and 1% for CO₂ in the presence of O₂ (20%). The enzyme is inhibitedby phenanthroline (PNT) (specific chelating agent of ferrousion), and competitively inhibited (K₁, =0.5 mM) by 2–aminoisobutyricacid (AIB), and the enzymatic activity was not detectable inthe absence of CO₂. Under optimal assay conditions, the enzymehas two optimum temperatures (28 C and 35 C) and is inhibitedby divalent metal cations (Zn²⁺> CO²⁺>Ni²⁺>Cu²⁺>Mn²⁺>Mg²⁺) and by salicylic acid, propylgallate, carbonyl cyanidem–chlorophenyl hydrazone (CCCP), dinitrophenol (DNP),and Na–benzoate. The in vitro ACCO activity which we recoveredin soluble form is equivalent to approximately 80–85%of the apparent activity evaluated in vivo. Key words: ACC oxidase, Cicer arietinum, ethylene, germination, seeds     

Further Characteristics of Salt-Dependent Bicarbonate Use by the Seagrass Zostera muelleri

Millhouse, J. and Strother, S. 1987. Further characteristicsof salt-dependent bicarbonate use by the seagrass Zostera muelleri.—J.exp. Bot. 38: 1055–1068. The contribution of HCO^–₃to photosynthetic O₂ evolutionin the seagrass Zostera muelleri Irmisch ex Aschers. increasedwith increasing salinity of the bathing seawater when the inorganiccarbon concentration was kept constant. K_1/2 (seawater salts)for HCO^–₃ -dependent photosynthesis was 66% of seawatersalinity. Both short- and long-term pretreatment at low salinitiesstimulated photosynthesis in full strength seawater. Twentyfour hours pre-incubation of seagrass plants in 3·0 molm^–3 NaHCO₃ resulted in increased photosynthesis at allsalinities, apparently due to stimulation of HCO^–₃ use(K_1/2 (seawater salts) = 26%). V_max (HCO^–₃) was not affectedby low salinity pretreatment. The kinetics of HCO^–₃ stimulationby the major seawater cations was investigated. Ca²⁺ was themost effective cation with the highest V_max (HCO^–₃) andwith K_1/2(Ca²⁺) = 14 mol m^–3. Mg²⁺ was also very effectiveat less than 50 mol m^–3 but higher concentrations wereinhibitory. This inhibition cannot be accounted for solely byprecipitation of MgCO₃. Na⁺ and K⁺ were both capable of stimulatingHCO^–₃ use. Stimulation was in two distinct parts. Up to500 mol m^–3, both citrate and chloride salts gave similarresults (K_1/2(Na⁺) 81 mol m^–3, V_max(HCO^–₃) 0·26µmol O₂ mg^–1 chl min^–1), but use of citratesalts above 500 mol m^–2 caused a second stimulation ofHCO^–₃ use (K_1/2(Na⁺) 830 mol m^–3, V_max(HCO^–₃)0·68 µmol O₂ mg^–1 chl min^–1). V_max(HCO^–₃)for the second-phase Na⁺ or K⁺ stimulation was of the same orderas for Ca²⁺-stimulated HCO^–₃ use. To further characterizesalt-dependent HCO^–₃ use, the sensitivity of photosynthesisto Tris and TES buffers was investigated. The effects of Trisappear to be due to the action of Tris⁺ causing stimulationof HCO^–₃ -dependent photosynthesis in the absence of salt,but inhibition of HCO^–₃ use in saline media. TES has noeffect on photosynthesis. External carbonic anhydrase, althoughimplicated in salt-dependent HCO^–₃ use in Z. muelleri,could not be detected in whole leaves. Key words: Zostera muelleri, HCO^–₃ use, salinity     

Regulation of Pyruvate Decarboxylase In Vitro and In Vivo

Results presented in this paper strongly support the view thatregulation of the key enzyme of alcoholic fermentation, pyruvatedecarboxylase (PDC), is achieved in a number of ways, all associatedwith possible lowering of the cytoplasmic pH during anoxia.These mechanisms include not only the well-known acid pH optimumof PDC, but also long-term, reversible changes in characteristicsof the enzyme established both in vitro and in vivo. Following transfer of desalted extracts from pH 6.0 to 7.4,maximal activity of PDC was decreased, while there was a considerableincrease in the lag before maximal activity was reached. Similarchanges in enzyme characteristics were observed when wheat (Triticumaestivum L. cv. Gamenya) roots and rice (Oryza sativa L. cv.Calrose) coleoptiles were transferred from anoxic to aerobicsolutions, provided PDC was assayed within 10 min of the startof maceration. All of the above changes were usually readilyreversible when extracts were returned to pH 6.0, or when plantswere returned to anoxic solutions. Additional regulation of PDC would be achieved by the S_0.5 forpyruvate which is 0.75 mol m^–3 at pH 6.0, 1.0 mol m^–3at pH 6.8, and 2.5 mol m^–3 at pH 7.4; the latter is wellabove estimates for pyruvate concentrations in the cytoplasmof aerated tissues. We assess that the combined effects of the acid pH optimum,the high S_0.5 at pH 7.4 and the long-term decreases in activityobserved during incubation at pH 7.4 would reduce PDC activityin aerobic cells to at most 7% of the activity in anoxic cells.Possible additional controls for the pathway of alcoholic fermentationare briefly considered. Key words: PDC, regulation, anoxia     

Stimulatory Effect of Chloride Ions on NADP Malic Enzyme from Leaves of two C4 Species of Cyperus

NADP malic enzyme (EC 1.1.1.40 [EC] ) from leaves of two C₄ speciesof Cyperus (C. rotundus and C. brevifolius var leiolepis) exihibiteda low level of activity in an assay mixture that contained lowconcentrations of Cl^–. This low level of activity wasmarkedly enhanced by increases in the concentration of NaClup to 200 mM. Since the activity of NADP malic enzyme was inhibitedby Na₂SO₄ and stimulated by relatively high concentration ofTris-HCl (50–100 mM, pH 7–8), the activation ofthe enzyme by NaCl appears to be due to Cl^–. Variationsin the concentration of Mg²⁺ affected the K_A (the concentrationof activator giving half-maximal activation) for Cl^–,which decreased from 500 mM to 80 mM with increasing concentrationsof Mg²⁺ from 0.5 mM to 7 mM. The K_m for Mg²⁺ was decreased from7.7 mM to 1.3 mM with increases in the concentration of NaClfrom zero to 200 mM, although the increase of V_max was not remarkable.NADP malic enzyme from Cyperus, being similar to that from otherC₄ species, was able to utilize Mn²⁺. The K_m for Mn²⁺ was 5mM, a value similar to that for Mg²⁺. The addition of 91 mMNaCl markedly decreased the K_m for Mn²⁺ to 20 +M. NADP malicenzyme from Setaria glauca, which contains rather less Cl^–than other C₄ species, was inactivated by concentrations ofNaCl above 20 mM, although slight activation of the enzyme wasobserved at low concentrations of NaCl at pH7.6. (Received February 20, 1989; Accepted June 12, 1989)     

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)     

Inhibition of Nodule Development in Soybean by Nitrate or Reduced Nitrogen

Imsande, J. 1986. Inhibition of nodule development in soybeanby nitrate or reduced nitrogen.—J. exp. Bot. 37: 348–355. Nodulation of hydroponically grown soybean plants [Glycine max(L.) Merr.] is inhibited by continuous growth in the presenceof 4· mol m^–3 KNO₃ The presence of 4·0 molm^–3 ‘starter nitrate’ for 3-6 d during noduledevelopment, however, subsequently stimulates nodule dry weightaccumulation and nitrogenase activity. These stimulations occureven though 4· mol m^–3 nitrate temporarily delaysnodule development, i.e. the late steps of nodule developmentare reversibly inhibited by a short-term exposure to 4·0mol m^–3 nitrate. On the other hand, treatment with 4·0mol m^–3 nitrate in excess of 14 d significantly reducesnodule dry weight Thus, extended growth in the presence of 4·0mol m^–3 KNO₃ seems to block both early and late stepsof nodule development. Nodulation of hydroponically grown soybeansis also inhibited by continuous growth in the presence of 2·0mol m^–3 (NH₄)₂SO₄ This inhibition is not caused by acidityof the growth medium. On the other hand, nodule development6 d after inoculation with Rhizoblum japonicum is not delayedby a 7-d exposure to 2·0 mol m^–3 (NH₄)₂SO₄ butis partially inhibited by a prolonged exposure to (NH₄)₂SO₄Because repression of nodulation by 4·0 mol m^–3KNO₃ is more severe than that by 2·0 mol m^–3 (NH₄)₂SO₄and because ammonium taken up by the soybean plant is not activelyoxidized to nitrate, it is suggested that there are at leasttwo mechanisms by which nitrate utilization represses noduleformation in soybean. Key words: Glycine max, nitrogen, nitrogen fixation, nodulation     

Plant Growth in Relation to the Supply and Uptake of NO3-: A Comparison Between Relative Addition Rate and External Concentration as Driving Variables

Two approaches to quantifying relationships between nutrientsupply and plant growth were compared with respect to growth,partitioning, uptake and assimilation of NO₃^– by non-nodulatedpea (Pisum sativum L. cv. Marma). Plants grown in flowing solutionculture were supplied with NO₃^– at relative addition rates(RAR) of 0·03, 0·06, 0·12, and 0·18d^–1, or constant external concentrations ([NO₃^–)of 3, 10, 20, and 100 mmol m^–3 over 19 d. Following acclimation,relative growth rates (RGR)approached the corresponding RARbetween 0·03–0.12 d^-1, although growth was notlimited by N supply at RAR =0.18 d^-1. Growth rates showed littlechange with [NO₃–] between 10–100 mmol m^–3(RGR=0·15 –0·16 d^-1). The absence of growthlimitation over this range was suggested by high unit absorptionrates of NO₃^–, accumulation of NO₃^– in tissues andprogressive increases in shoot: root ratio. Rates of net uptakeof NO₃^– from 1 mol m^–3 solutions were assessed relativeto the growth-related requirement for NO₃^–, showing thatthe relative uptake capacity increased with RGR between 0·03–0·06d^–1 , but decreased thereafter to a theoretical minimumvalue at RGR