Flow cytometric and microscopic analysis of the effect of tannic acid on plant nuclei and estimation of DNA content

• Background and Aims Flow cytometry (FCM) is extensivelyused to estimate DNA ploidy and genome size in plants. In orderto determine nuclear DNA content, nuclei in suspension are stainedby a DNA-specific fluorochrome and fluorescence emission isquantified. Recent studies have shown that cytosolic compoundsmay interfere with binding of fluorochromes to DNA, leadingto flawed data. Tannic acid, a common phenolic compound, maybe responsible for some of the stoichiometric errors, especiallyin woody plants. In this study, the effect of tannic acid onestimation of nuclear DNA content was evaluated in Pisum sativumand Zea mays, which were chosen as model species. • Methods Nuclear suspensions were prepared from P. sativumleaf tissue using four different lysis buffers (Galbraith's,LB01, Otto's and Tris.MgCl₂). The suspensions were treated withtannic acid (TA) at 13 different initial concentrations rangingfrom 0·25 to 3·50 mg mL^–1. After propidiumiodide (PI) staining, samples were analysed using FCM. In additionto the measurement of nuclei fluorescence, light scatter propertieswere assessed. Subsequently, a single TA concentration was chosenfor each buffer and the effect of incubation time was assessed.Similar analyses were performed on liquid suspensions of P.sativum and Z. mays nuclei that were isolated, treated and analysedsimultaneously. FCM analyses were accompanied by microscopicobservations of nuclei suspensions. • Key Results TA affected PI fluorescence and light scatterproperties of plant nuclei, regardless of the isolation bufferused. The least pronounced effects of TA were observed in Tris.MgCl₂buffer. Samples obtained using Galbraith's and LB01 bufferswere the most affected by this compound. A newly described ‘tannicacid effect’ occurred immediately after the addition ofthe compound. With the exception of Otto's buffer, nuclei ofP. sativum and Z. mays were affected differently, with pea nucleiexhibiting a greater decrease in fluorescence intensity. • Conclusions A negative effect of a secondary metabolite,TA, on estimation of nuclear DNA content is described and recommendationsfor minimizing the effect of cytosolic compounds are presented.Alteration in light scattering properties of isolated nucleican be used as an indicator of the presence of TA, which maycause stoichiometric errors in nuclei staining using a DNA intercalator,PI.     

RNA Synthesis in Phaseolus Chloroplasts: I. RIBONUCLEIC ACID SYNTHESIS IN CHLOROPLAST PREPARATIONS FROM PHASEOLUS VULGARIS L. LEAVES AND SOLUBILIZATION OF THE RNA POLYMERASE

Chloroplast preparations from the young primary leaves of Phaseolusvulgaris L. cv. Canadian Wonder carry out the DNA-dependentincorporation of UTP into RNA at rates between 8 and 14 pmolUTP µg^–1 chlorophyll h^–1. It is estimatedthat 90% of the activity was localized in the chloroplasts.The incorporation proceeded for between 20 and 30 min at 35°C. The maximum rates of RNA synthesis were attained atpH 8.3, in the presence of 15 mM MgCl₂. Chloroplasts were alsoactive, to a lesser extent, with 1.5 mM MnCl₂. The simultaneouspresence of MnCl₂ and MgCl₂ resulted in inhibition of activity.Nuclear material prepared from young P. vulgaris leaves incorporatedUTP at a rate of about 12 pmol UTP µg^–1 DNA h^–1.On a chloroplast (Tritonsoluble) DNA basis chloroplast activitywas over 40-fold that of nuclei. Methods of solubilizing chloroplastRNA polymerase were explored. Yields of over 75% were achieved,but methods suitable for one species were not always successfulwhen applied to another. The highest yields of the P. vulgarisenzyme were obtained using EDTA and KCl. All methods resultedin solubilization of DNA. RNA synthesis by the soluble P. vulgarisenzyme proceeded for more than 40 min at 35 °C.     

Nutrient Solution pH Control using Dipolar Buffers in Studies of Trifolium repens L. Nitrogen Nutrition

In studies of Trifolium repens nitrogen nutrition, the controlof nutrient solution pH using dipolar buffers, was evaluatedin tube culture under sterile conditions. Five buffers; MES,ADA, ACES, BES and MOPS with pK₂s (20 °C) of 6.15, 6.60,6.90, 7.15 and 7.20 respectively, at a concentration of 2.0mol m^–3, were provided to inoculated Trifolium repensgrowing in nutrient solution containing 7.13 mol m^–3 nitrogenas (NH₄)₂SO₄. Initial pH of each solution was adjusted to theappropriate buffer pK₂ Two buffers, ADA and ACES completelyinhibited plant growth. The remaining buffers had little effectin limiting pH change, although plant dry matter was higherand nodule numbers lower in the presence of these buffers. MESand MOPS were supplied to nutrient solutions with and without7.13 mol m^–3 (NH₄)₂SO₄, at concentrations ranging from0–12 mol m^–3. MES at 9 mol m^–3 and 12 molm^–3 reduced growth of plants reliant on the symbiosisfor providing nitrogen. The provision of MES to plants providedwith NH₄⁺ significantly increased plant yield and reduced nodulenumber at all concentrations. MOPS did not affect plant yieldor nodule number. The use of dipolar buffers in legume nitrogennutrition studies is considered in terms of buffering capacity,and the side effects on plant growth and symbiotic development. Key words: Ammonium, Dipolar buffer, Nitrogen nutrition, pH control, Symbiosis, Trifolium repens     

Genetic diversity and geographic differentiation in endangered Ammopiptanthus (Leguminosae) populations in desert regions of northwest China as revealed by ISSR analysis

• Background and Aims The desert legume genus Ammopiptanthuscomprises two currently endangered species, A. mongolicus andA. nanus. Genetic variability and genetic differentiation betweenthe two species and within each species were examined. • Methods Inter-simple sequence repeat (ISSR) marker datawere obtained and analysed with respect to genetic diversity,structure and gene flow. • Key Results Despite the morphological similarity betweenA. mongolicus and A. nanus, the two species are geneticallydistinct from each other, indicated by 63 % species-specificbands. Low genetic variability was detected for both populationlevel (Shannon indices of diversity H_pop = 0·106, percentageof polymorphic loci P = 18·55 % for A. mongolicus; H_pop= 0·070, P = 12·24 % for A. nanus) and specieslevel (H_sp = 0·1832, P = 39·39 % for A. mongolicus;H_sp = 0·1026, P = 25·89 % for A. nanus). Moderategenetic differentiation was found based on different measures(AMOVA _ST and Hickory ^B) in both A. mongolicus (0·3743–0·3744)and A. nanus (0·2162–0·2369). • Conclusions The significant genetic difference betweenthe two species might be due to a possible vicariant evolutionaryevent from a single common ancestor through the fragmentationof their common ancestor's range. Conservation strategies forthese two endangered species are proposed.     

Anion-dependent changes of energy transfer in chloroplasts II. Relationships of the coupling factor to light-induced proton transport and absorbance change at 515 nm

Roles of the coupling factor in light-induced proton transportand 515-nm absorption change were investigated in chloroplastswashed with high concentrations of Tris salts (pH 7.2). Washingthe chloroplasts with Tris-HCl and Tris-HNO₃ buffers diminishedboth the light-induced pH rise and absorbance change at 515-nm,while Tris-H₂SO₄ buffer was much less effective. Inhibited activitiescould be restored by replacement of the coupling factor afterextraction with EDTA. N,N'-dicyclohexylcarbodiimide also restoredboth activities. Effects of various anions on the proton pumpand 515-nm shift were also investigated. The order of effectivenesswas NO₃^–>Cl^–>SO₄^2–. The role of thecoupling factor and its mode of action; the action mechanismsof Tris and anionsn energy transducing processes in chloroplasts,photophosphorylation, proton transport and absorbance changeat 515 nm, are discussed. ¹Present address: Biology Department, College of Science andEngineering, Ryukyu University, Naha, Okinawa, Japan. (Received June 27, 1972; )     

Photosynthetic and Photorespiratory Enzymes in Widely Divergent Plant Species with Special Reference to the Moss Ceratodon purpureus: Properties of Ribulose Bisphosphate Carboxylase/ Oxygenase, Phosphoenolpyruvate Carboxylase and Glycolate Oxidase

Rintamäki, E. and Aro, E.-M. 1985. Photosynthetic and photorespiratoryenzymes in widely divergent plant species with special referenceto the moss Ceratodon purpureus: Properties of ribulose bisphosphatecarboxylase/oxygenase, phosphoenolpyruvate carboxylase and glycolateoxidase.—J. exp. Bot. 36: 1677–1684. Km(CO₂) values and maximal velocities of ribulose bisphosphatecarboxylase/oxygenase (E.C. 4.1.1.39 [EC] ) were determined for sixplant species growing in the wild, consisting of a moss, a fernand four angiosperms. The maximum velocities of the RuBP carboxylasesvaried from 0.13 to 0.;62 µmol CO₂ fixed min^–1 mg^–1soluble protein and the Km(CO₂) values from 15 to 22 mmol m^–3CO₂. The highest Km(CO₂) values found were for the moss, Ceratodonpurpureus, and the grass, Deschampsia flexuosa. These plantsalso had the highest ratios of the activities of RuBP carboxylaseto RuBP oxygenase. Glycolate oxidase (E.C. 1.1.3.1 [EC] ) activitieswere slightly lower in D.flexuosa, but not in C. purpureus,than for typical C₃ species. Phosphoenolpyruvate carboxylase(E.C. 4.1.1.31 [EC] ) was not involved in the photosynthetic carboxylationby these two plants. However, another grass, Phragmites australis,was intermediate in PEP carboxylase activity between C₃ andC₄ plants The properties of RuBP carboxylase/oxygenase are discussedin relation to the activities of PEP carboxylase and glycolateoxidase and to the internal CO₂ concentration. Key words: RuBP carboxylase, oxygenase, Km(CO₂), moss     

Acid phosphatase role in chickpea/maize intercropping

• Background and aims Organic P comprises 30–80 %of the total P in most agricultural soils. It has been proventhat chickpea facilitates P uptake from an organic P sourceby intercropped wheat. In this study, acid phosphatase excretedfrom chickpea roots is quantified and the contribution of acidphosphatase to the facilitation of P uptake by intercroppedmaize receiving phytate is examined. • Methods For the first experiment using hydroponics, maize(Zea mays ‘Zhongdan No. 2’) and chickpea (Cicerarietinum ‘Sona’) were grown in either the sameor separate containers, and P was supplied as phytate, KH₂PO₄at 0·25 mmol P L^–1, or not at all. The second experimentinvolved soil culture with three types of root separation betweenthe two species: (1) plastic sheet, (2) nylon mesh, and (3)no barrier. Maize plants were grown in one compartment and chickpeain the other. Phosphorus was supplied as phytate, Ca(H₂PO₄)₂at 50 mg P kg^–1, or no P added. • Key results In the hydroponics study, the total P uptakeby intercropped maize supplied with phytate was 2·1-foldgreater than when it was grown as a monoculture. In the soilexperiment, when supplied with phytate, total P uptake by maizewith mesh barrier and without root barrier was 2·2 and1·5 times, respectively, as much as that with solid barrier.In both experiments, roots of both maize and chickpea suppliedwith phytate and no P secreted more acid phosphatase than thosewith KH₂PO₄ or Ca(H₂PO₄)₂. However, average acid phosphataseactivity of chickpea roots supplied with phytate was 2–3-foldas much as maize. Soil acid phosphatase activity in the rhizosphereof chickpea was also significantly higher than maize regardlessof P sources. • Conclusions Chickpea can mobilize organic P in both hydroponicand soil cultures, leading to an interspecific facilitationin utilization of organic P in maize/chickpea intercropping.     

Affinity of RuBP Carboxylases for Carbon Dioxide and Inhibition of the Enzymes by Oxygen

Ribulose bisphosphate carboxylase (E.C.4.1.1.39) was purifiedfrom leaves of Triticum aestivum, Hordeum vulgare, Spinaceaoleracea, Petroselinum crispum, salad mustard-most likely Brassicanapus, Helianthus annuus, Solanum tuberosum, Beta vulgaris,Lolium perenne, Equisetum arvense, Zea mays, Ginkgo biloba,Pteris aquilina, Salix babylonica, Chamaecyparis lawsonianaand Atrichum undulatum by density gradient centrifugation andgel filtration or by ammonium sulphate fractionation, densitygradient centrifugation, ion-exchange chromatography and gelfiltration. Purified enzymes were freeze-dried and then storedat 0 °C to 4 °C. Portions of each enzyme preparationwere reactivated at 25 °C for 5 h in the presence of 10mM HCO₂ and 20 mM MgCl₂-RuBP carboxylase activities were measuredat four different concentrations of CO₂ at 25 °C and pH8.2 in solutions equilibrated with pure nitrogen or air (21%O₂, 79% N₂). K_m(CO₂), V_max and K₁(O₂) values were computed fromthe results. Significant differences were found in the K_m(CO₂)values for enzymes isolated from different species. Sensitivityof the enzymes to oxygen was less variable.     

Effects of the fungal endophyte, Neotyphodium lolii, on net photosynthesis and growth rates of perennial ryegrass (Lolium perenne) are independent of In Planta endophyte concentration

• Background and Aims Neotyphodium lolii is a fungal endophyteof perennial ryegrass (Lolium perenne), improving grass fitnessthrough production of bioactive alkaloids. Neotyphodium speciescan also affect growth and physiology of their host grasses(family Poaceae, sub-family Pooideae), but little is known aboutthe mechanisms. This study examined the effect of N. lolii onnet photosynthesis (P_n) and growth rates in ryegrass genotypesdiffering in endophyte concentration in all leaf tissues. • Methods Plants from two ryegrass genotypes, Nui D andNui UIV, infected with N. lolii (E+) differing approx. 2-foldin endophyte concentration or uninfected clones thereof (E–)were grown in a controlled environment. For each genotype xendophyte treatment, plant growth rates were assessed as tilleringand leaf extension rates, and the light response of P_n, darkrespiration and transpiration measured in leaves of young (30–45d old) and old (>90 d old) plants with a single-chamber openinfrared gas-exchange system. • Key Results Neotyphodium lolii affected CO₂-limited ratesof P_n, which were approx. 17 % lower in E+ than E– plants(P < 0·05) in the young plants. Apparent photon yieldand dark respiration were unaffected by the endophyte (P >0·05). Neotyphodium lolii also decreased transpiration(P < 0·05), but only in complete darkness. There wereno endophyte effects on P_n in the old plants (P > 0·05).E+ plants grew faster immediately after replanting (P < 0·05),but had approx. 10 % lower growth rates during mid-log growth(P < 0·05) than E– plants, but there was noeffect on final plant biomass (P > 0·05). The endophyteeffects on P_n and growth tended to be more pronounced in NuiUIV, despite having a lower endophyte concentration than NuiD. • Conclusions Neotyphodium lolii affects CO₂ fixation,but not light interception and photochemistry of P_n. The impactof N. lolii on plant growth and photosynthesis is independentof endophyte concentration in the plant, suggesting that theendophyte mycelium is not simply an energy drain to the plant.However, the endophyte effects on P_n and plant growth are stronglydependent on the plant growth phase.     

Modelling environmental variation in Young's modulus for Pinus radiata and implications for determination of critical buckling height

• Background and Aims Although density-specific stiffness,E/, (where E is Young's modulus and is wood density) is oftenassumed constant by the elastic similarity model, and in determinationof critical buckling height (H_crit), few studies have testedthis assumption within species. Here this assumption is testedfor Pinus radiata growing across an environmental gradient,and theory is combined with data to develop a model of Young'smodulus. • Methods Analyses use an extensive series of environmentalplots covering the range of climatic and edaphic conditionsover which P. radiata is grown in New Zealand. Reduced majoraxis regression was used to determine scaling exponents betweenlog–log plots of H_crit vs. groundline diameter (D), andE/ vs. D. Path analysis was used to identify significant directand indirect (through stem slenderness) edaphic and climaticinfluences on E. • Key Results Density-specific stiffness exhibited 3-foldvariation. As E/ scaled positively with D, the exponent of 0·95between H_crit and D exceeded the assumed value of 0·67under constant E/. The final path analysis model included meanair temperature in early autumn (T_aut) and slenderness as significant(P < 0·05) positive direct influences on E. Tree leafarea index and T_aut were indirectly associated with E throughtheir significant (P < 0·05) positive direct relationshipwith stem slenderness. Young's modulus was most sensitive toT_aut, followed by stem slenderness then leaf area index, andthe final model explained 76 % of the variance in E. • Conclusions The findings suggest that within speciesE/ variation may influence H_crit and the scaling exponent betweenD and H_crit so important in assumptions regarding allometricrelationships. The model presented may provide a useful meansof determining variation in E, E/ and H_crit across environmentalgradients.     

Effect of Light and NaCI Salinity on the Growth of Callus Cultures of Ipomoea pes-caprae (L.) R. Brown and Ipomoea batatas (L.) Lam

Callus cultures of Ipomoea pes-caprae and I. batatas were establishedon MS medium containing 10^–5 M 2,4-D and 10^–8 Mbenzyladenine. Ipomoea pes-caprae calli exhibited green pigmentationand grew better in the light than in darkness. Callus tissuesof I. batatas showed a pale-yellow colouration and they grewat the same rate in light as in dark conditions. I. pes-capraeand I. batatas callus cultures were sensitive to the presenceof 60 mM NaCl in the culture medium, the growth of the formerbeing more sensitive in light than in darkness. The significanceof the responses of I. pes-caprae callus cultures in relationto the mechanism of salt tolerance is discussed. Ipomoea batatas, Ipomoea pes-caprae, sweet potato, railroad vine, callus cultures, salinity, light     

The Effects of Cell Cycle Parameters on Cell Wall Regeneration and Cell Division of Cotton Protoplasts (Gossypium hirsutum L.)

Protoplasts of cotton cotyledons were isolated and culturedto undergo cell wall regeneration and cell division. DNA contentand cell cycle parameters of nuclei from cotyledons and/or protoplastswere determined by flow cytometry. The DNA content of cotton,Gossypium hirsutum L., was estimated to be 4·34±0·12pg DNA per nucleus. There was a strong positive correlation between G₂ or Sand G₂,and cell wall regeneration and cell division and a strong negativecorrelation between G₁, and cell wall regeneration and celldivision of cotton cotyledon protoplasts. The cell cycle statusof cotyledons changes during their development; as the cotyledonsenlarge, the proportion of cells in G₀ and G₁ phases of thecell cycle increases. The implication of these results in relationto protoplast growth and development is discussed. Key words: Cell cycle parameters, cell wall regeneration, cell division, flow cytometry, Gossypium     

Effects of Calcium and Potassium Ions on Phototaxis in Cryptomonas

Effects on positive phototaxis and the cell motility of 7 cationsin 5mM MOPS (morpholinopropane sulfonic acid) buffer (pH 7.0)containing 0.16 mM NaCl, 0.68 mM KCl, 0.5 mM CaCl₂ and 0.16mM MgCl₂ were studied in the unicellular flagellate Cryptomonaswith a photoelectrical measuring apparatus and photomicrography.When calcium ion was removed from the medium by adding 1 mMEGTA (ethylene glycol-bis-(ß-amino-ethylether)-N,N'-tetraaceticacid), the phototactic response was totally inhibited, but theswimming rate was not much affected. The effect of EGTA waspartially reversed by the addition of 1 mM CaCl₂. When 15mMKCl or RbCl was added to the medium, phototaxis was greatlyinhibited, but there was no significant influence on the swimmingrate. Similar but less inhibitory effects were induced in thepresence of NaCl, LiCl and CsCl. KCl-induced inhibition waspartially removed by the addition of 15 mM CaCl₂ or MgCl₂. (Received June 25, 1982; Accepted September 27, 1982)     

Photosynthetic Characteristics of C3-C4 Intermediate Flaveria Species II. Kinetic Properties of Phosphoenolpyruvate Carboxylase from C3, C4 and C3-C4 Intermediate Species

The kinetic properties of phosphoenolpyruvate (PEP) carboxylasehave been studied among several Flaveria species: the C₃ speciesF. cronquistii, the C₃–C₄ species F. pubescens and F.linearis, and the C₄ species F. trinervia. At either pH 7 or8, the maximum activities (in µmol.mg Chl^–1.h^–1)for F. pubescens and linearis (187–513) were intermediateto those of the C₃ species (12–19) and the C₄ species(2,182–2,627). The response curves of velocity versusPEP concentration were hyperbolic for the C₃ and C₃–C₄species at either pH 7 or 8 while they were sigmoidal for theC₄ species at pH 7 and hyperbolic at pH 8. The K_m values forPEP determined from reciprocal plots were lowest in the C₃ species,and of intermediate value in the C₃–C₄ species comparedto the K' values of the C₄ species determined from Hill plotsat either pH 7 or 8. Glucose-6-phosphate (G6P) decreased theK_m values for PEP at both pH 7 and 8 in the C₃ and C₃–C₄species. In the C₄ species, G6P decreased the K' values at pH8 but increased the K' values at pH 7. In all cases, G6P hadits effect by influencing the activity at limiting PEP concentrationswith little or no effect on the maximum activity. At pH 8 andlimiting concentrations of PEP the degree of stimulation ofthe activity by G6P was greatest in the C₄ species, intermediatein F. linearis, a C₃–C₄ species, and lowest in the C₃species. In several respects, the PEP carboxylases of the C₃–C₄Flaveria species have properties intermediate to those of theC₃ and C₄ species. (Received April 30, 1983; Accepted August 22, 1983)     

A Comparison of the Growth, Photosynthesis, Stomatal Conductance and Water Use Efficiency of Moricandia and Brassica Species

When grown in pots and well-watered, the relative growth ratesof the above ground parts of two species of Moricandia (M. arvensis,an intermediate C₃–C₄ species, and M. moricandioides,a C₃ species) were inferior to those of two cultivated Brassicaspecies (B. campestris and B. napus). The Moricandia specieshad thicker leaves (greater d.wt per unit leaf area) with morechlorophyll than the Brassica species and had slightly greaterrates of photosynthesis per unit leaf area at an irradiance(400–700 nm) of 2000 µmol quanta m^–2 s ^–1.Leaves of M. arvensis, known to have a CO₂ compensation pointbetween that of C₃ and C₄ species, had a lower ratio of theintercellular to atmospheric partial pressure of CO₂ (C₁/C_a)and a greater instantaneous water use efficiency (WUE) thanthose of M. moricandioides and the Brassica species. Carbon isotope discrimination (     

Fine-scale genetic structure among genetic individuals of the clone-forming monotypic genus Echinosophora koreensis (Fabaceae)

• Background and Aims For rare endemics or endangered plantspecies that reproduce both sexually and vegetatively it iscritical to understand the extent of clonality because assessmentof clonal extent and distribution has important ecological andevolutionary consequences with conservation implications. Asurvey was undertaken to understand clonal effects on fine-scalegenetic structure (FSGS) in two populations (one from a disturbedand the other from an undisturbed locality) of Echinosophorakoreensis, an endangered small shrub belonging to a monotypicgenus in central Korea that reproduces both sexually and vegetativelyvia rhizomes. • Methods Using inter-simple sequence repeats (ISSRs) asgenetic markers, the spatial distribution of individuals wasevaluated using Ripley's L(d)-statistics and quantified thespatial scale of clonal spread and spatial distribution of ISSRgenotypes using spatial autocorrelation analysis techniques(join-count statistics and kinship coefficient, F_ij) for totalsamples and samples excluding clones. • Key Results A high degree of differentiation betweenpopulations was observed (_ST(g) = 0·184, P < 0·001).Ripley's L(d)-statistics revealed a near random distributionof individuals in a disturbed population, whereas significantaggregation of individuals was found in an undisturbed site.The join-count statistics revealed that most clones significantlyaggregate at 6-m interplant distance. The Sp statistic reflectingpatterns of correlograms revealed a strong pattern of FSGS forall four data sets (Sp = 0·072–0·154), butthese patterns were not significantly different from each other.At small interplant distances (2 m), however, jackknifed 95% CIs revealed that the total samples exhibited significantlyhigher F_ij values than the same samples excluding clones. • Conclusion The strong FSGS from genets is consistentwith two biological and ecological traits of E. koreensis: bee-pollinationand limited seed dispersal. Furthermore, potential clone matesover repeated generations would contribute to the observed highF_ij values among genets at short distance. To ensure long-termex situ genetic variability of the endangered E. koreensis,individuals located at distances of 10–12 m should becollected across entire populations of E. koreensis.     

Studies on the Growth in Culture of Plant Cells: XXIII ISOLATION OF NUCLEI AND HISTONES FROM CULTURED CELLS OF ACER PSEUDOPLATANUS L.

A technique is described for the isolation of purified nucleifrom suspension culture cells of Acer pseudoplatanus. This involvesa grinding medium containing 70% (v/v) glycerol, 1 mM Mg²⁺,2 mM Ca²⁺, and Tris buffer at pH 7.8, prestorage and disruptionof the cells at –20 °C in a Potter-Elvehjem homogenizer,and purification by filtration and centrifugation in the presenceof Triton X-100. The nuclear yield is c. 25% as assessed bynuclear count or DNA estimation and the nuclei are active inthe RNA synthesizing system of Tautvydas (1971). When the histones of these nuclei are extracted in H₂SO₄ andprecipitated by ethanol, 113 µg histone is obtained perµg nuclear DNA and the histone fraction contains 22% basicamino acids and has a lysine: arginine ratio of 2.6. Acid-ureagel electrophoresis shows the presence of five major histones(H1, H2A, H2B, H3, and H4 in sequence from anode to cathode)having respectively molecular weights of 24 500, 13 500, 13300, 12 800, and 11 000. There is very good correspondence betweencalf thymus histones H3 (reduced form) and H4 and two of theseAcer histones. The other Acer histones differ from the calfthymus histones H1, H2A, H2B in molecular weight but can beprovisionally equated with these by a newly developed differentialstaining reaction. Calf thymus histone H2A appears to be lessrich in lysine than the corresponding Acer histone. Evidence from a pulse-chase experiment with (¹⁴C)lysine and[³H]tryptophan is in favour of the cytoplasmic synthesis ofthe histones.     

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     

Plant allometry, leaf nitrogen and phosphorus stoichiometry, and interspecific trends in annual growth rates

• Background Life forms as diverse as unicellular algae,zooplankton, vascular plants, and mammals appear to obey quarter-powerscaling rules. Among the most famous of these rules is Kleiber's(i.e. basal metabolic rates scale as the three-quarters powerof body mass), which has a botanical analogue (i.e. annual plantgrowth rates scale as the three-quarters power of total bodymass). Numerous theories have tried to explain why these rulesexist, but each has been heavily criticized either on conceptualor empirical grounds. • N,P-Stoichiometry Recent models predicting growth rateson the basis of how total cell, tissue, or organism nitrogenand phosphorus are allocated, respectively, to protein and rRNAcontents may provide the answer, particularly in light of theobservation that annual plant growth rates scale linearly withrespect to standing leaf mass and that total leaf mass scalesisometrically with respect to nitrogen but as the three-quarterspower of leaf phosphorus. For example, when these relationshipsare juxtaposed with other allometric trends, a simple N,P-stoichiometricmodel successfully predicts the relative growth rates of 131diverse C₃ and C₄ species. • Conclusions The melding of allometric and N,P-stoichiometrictheoretical insights provides a robust modelling approach thatconceptually links the subcellular ‘machinery’ ofprotein/ribosomal metabolism to observed growth rates of uni-and multicellular organisms. Because the operation of this ‘machinery’is basic to the biology of all life forms, its allometry mayprovide a mechanistic explanation for the apparent ubiquityof quarter-power scaling rules.     

Biochemical Limitations to Carbon Assimilation in C3 Plants--A Retrospective Analysis of the A/Ci Curves from 109 Species

Species-specific differences in the assimilation of atmosphericCO₂ depends upon differences in the capacities for the biochemicalreactions that regulate the gas-exchange process. Quantifyingthese differences for more than a few species, however, hasproven difficult. Therefore, to understand better how speciesdiffer in their capacity for CO₂ assimilation, a widely usedmodel, capable of partitioning limitations to the activity ofribulose-1,5-bisphosphate carboxylase-oxygenase, to the rateof ribulose 1,5-bisphosphate regeneration via electron transport,and to the rate of triose phosphate utilization was used toanalyse 164 previously published A/C_i, curves for 109 C₃ plantspecies. Based on this analysis, the maximum rate of carboxylation,Vc_max, ranged from 6µmol m^–2 s^–1 for the coniferousspecies Picea abies to 194µmol m^–2 s^–1 forthe agricultural species Beta vulgaris, and averaged 64µmolm^–2 s^–1 across all species. The maximum rate ofelectron transport, J_max, ranged from 17µmol m^–2s^–1 again for Picea abies to 372µmol m^–2 s^–1for the desert annual Malvastrum rotundifolium, and averaged134µmol m^–2 s^–1 across all species. A strongpositive correlation between Vc_max and J_max indicated that theassimilation of CO₂ was regulated in a co-ordinated manner bythese two component processes. Of the A/C_i curves analysed,23 showed either an insensitivity or reversed-sensitivity toincreasing CO₂ concentration, indicating that CO₂ assimilationwas limited by the utilization of triose phosphates. The rateof triose phosphate utilization ranged from 4·9 µmolm^–2 s^–1 for the tropical perennial Tabebuia roseato 20·1 µmol m^–2 s^–1 for the weedyannual Xanthium strumarium, and averaged 10·1 µmolm^–2 s^–1 across all species. Despite what at first glance would appear to be a wide rangeof estimates for the biochemical capacities that regulate CO₂assimilation, separating these species-specific results intothose of broad plant categories revealed that Vc_max and J_maxwere in general higher for herbaceous annuals than they werefor woody perennials. For annuals, Vc_max and J_max averaged 75and 154 µmol m^–2 s^–1, while for perennialsthese same two parameters averaged only 44 and 97 µmolm^–2 s^–1, respectively. Although these differencesbetween groups may be coincidental, such an observation pointsto differences between annuals and perennials in either theavailability or allocation of resources to the gas-exchangeprocess. Key words: A/C_i curve, CO₂ assimilation, internal CO₂ partial pressure, photosynthesis