Selection of Copper and Zinc Resistant Nicotiana plumbaginifolia Cell Suspension Cultures

Copper and zinc resistant cells of Nicotiana plumbaginifoliawere selected using unmutagenized cell suspensions in mediumcontaining normally lethal concentrations of CuSO₄ or ZnSO₄.Both resistances were retained for thirty cell doublings withoutselection pressure. The Cu resistant cells were 10-times andthe Zn resistant cells were 6-times as resistant as the wildtype cells. The Zn resistant cells were also somewhat resistantto AlCl₃ in comparison with the wild type cells, while the Curesistant cells were also somewhat resistant to ZnSO₄ and AlCl₃.The uptake of Cu by the Cu resistant cells and Zn by the Znresistant cells was higher than that of the wild type cells. (Received April 21, 1986; Accepted June 30, 1986)     

Translocation of copper and other micronutrients in tomato plants (Lycopersicon esculentum Mill.): nicotianamine-stimulated copper transport in the xylem

The influence of the endogenous micronutrient chelator, nicotianamine(NA), and of Cu nutrition on the distribution of Cu, Fe, Mn,Zn, and NA was investigated in eight different shoot organs,roots, and in xylem exudates of the NA-containing tomato wildtype Lycopersicon esculentum Mill. cv. Bonner Beste and itsNA-less mutant chloronerva. Contrary to the other heavy metals, copper transport in thexylem was inefficient in the mutant and was enhanced by an applicationof NA to the roots or leaves in proportion to the applied NAconcentration. Also, with NA application, the Cu concentrationin mutant roots decreased significantly, and increased in theshoot. Fe and Mn transport in the xylem was greater in the mutantthan in the wild type, and was decreased in the mutant by theapplication of NA to the leaves. Zn transport in the xylem wasthe same in both genotypes and was unaffected by NA application.After application of NA to leaves and roots of the mutant itwas possible to detect NA in the xylem exudate (up to 2nmolNA(g^–1 root FWh^–1). High Cu supply (3 µM) resulted in higher Cu and Mn concentrationsin all organs of the wild type as compared to mutant organs,but Fe concentrations were not influenced. Under high Cu supply(3µM) the NA concentrations of roots and the three youngestleaves of the wild type were higher than under normal Cu supply(0.3 µM). The highest concentrations were found in theshoot apex under both Cu conditions (up to 361 nmol NAg^–1FW). It is concluded from our experiments and from the high stabilityconstant of the NA-Cu-complex (log K= 18.6) that NA is involvedin Cu translocation whereas for the translocation of Fe, Mn,and Zn, NA is not essential. Key words: Copper transport, micronutrients, mobilization, nicotianamine, xylem     

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.     

The Movement of Zinc through Excised Stems of Seedlings of Pinus radiata D. Don.

Radioactive Zn solns were drawn through 10 cm stem sectionsexcised from seedlings of P. radiata to determine the locationand quantity of ⁶⁵Zn remaining in the stems and the concn of⁶⁵Zn in the exudate. Zinc was removed from solns passing through the excised stemsby processes which appeared to be non-metabolic because coolingthe inflow solns did not decrease the proportion of Zn removedand non-specific because Ca competed with Zn for ‘exchangesites’. The formation of anionic or uncharged complexes between Zn andEDTA, or citrate resulted in more Zn passing through the excisedstems. Consistent with the greater stability of Zn-EDTA complexesmore Zn passed through stems treated with EDTA than with citrate.Decreasing the pH of solns containing Zn, Ca and potassium citrateincreased the amount of Zn deposited in the basal (inflow) endof the stems, probably by decreasing the amount of Zn boundto citrate. Increasing the conen of Zn in test solns containingZn, Ca and potassium citrate did not change the distributionof ⁶⁵Zn in the stems as the capacity of the stems to removeZn from soln was large enough to remove all the free Zn in allthe solns Pinus radiata D. Don, pine, zinc, movement, stems     

Accumulation of Pb and Zn in Gametophytes and Sporophytes of the Moss Funaria hygrometrica(Funariales)

Accumulation of lead and zinc was studied in the moss Funariahygrometrica Hedw. collected from mine tailings. Heavy metalaccumulation in gametophytes and sporophytes was quantifiedby graphite furnace atomic absorption spectrometry (GFAAS) andinductively coupled plasma-atomic emission spectrometry (ICP-AES).Pb and Zn accumulation in the placental zone was analysed byx-ray scanning electron microscopy (SEM) and transmission electronmicroscopy (TEM) microanalysis. Spectrometry showed that whilemoss gametophytes accumulated considerable concentrations ofheavy metals, sporophytes accumulated only small concentrationsof metals. X-ray SEM and TEM showed that the two metals accumulatedin placental transfer cells on both the gametophytic and sporophyticsides. To investigate the uptake pattern for both metals undercontrolled conditions, F. hygrometrica plants collected froma non-polluted site were treated in the laboratory with separatesolutions of Pb and Zn at two concentrations (10^-2and 10^-4 M)for 24 or 168 h. Metal accumulation was analysed separatelyin gametophytes and sporophytes using GFAAS and ICP–AES.Each generation had a different accumulation quotient for bothmetals, and gametophytes accumulated significantly more metalthan sporophytes. Concentrations of Zn in sporophytes were alwayshigher than concentrations of Pb. The findings are discussedin relation to the role performed by the gametophyte and theplacenta in the accumulation and sequestration of Pb and Zn.Copyright 2001 Annals of Botany Company Atomic spectroscopy, Funaria hygrometrica, gametophyte, Pb and Zn accumulation, sporophyte, x-ray TEM and SEM microanalysis     

Malate Decarboxylation by Mitochondria of the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum

Mitochondria isolated from leaves of Mesembryanthemum crystallinumoxidized malate by both NAD malic enzyme and NAD malate dehydrogenase.Rates of malate oxidation were higher in mitochondria from plantsgrown at 400 mil NaCl in the rooting medium and performing Crassulaceanacid metabolism (CAM) than in mitochondria from plants grownat 20 mM NaCl and exhibiting C₃-photosynthetic CO₂ fixation.The mitochondria isolated from plants both in the CAM and C₃modes were tightly coupled and gave high respiratory control.At optimum pH for malate oxidation (pH 7.0), pyruvate was themajor product in mitochondria from CAM-M. crystallinum, whereasmitochondria from C₃-M. crystallinum produced predominantlyoxaloacetate. Both the extracted NAD malic enzyme in the presenceof CoA and the oxidation of malate to pyruvate by the mitochondriafrom plants in the CAM mode had a pH optimum around 7.0 withactivity declining markedly above this pH. The activity of NAD-malicenzyme, expressed on a cytochrome c oxidase activity basis,was much higher in mitochondria from the CAM mode than the C₃mode. The results indicate that mitochondria of this speciesare adapted to decarboxylate malate at high rates during CAM. ¹Current address: Lehrstuhl für Botanik II, UniversitätWurzburg, Mittlerer Dallenbergweg 64, 8700 Würzburg, WestGermany. ²Current address: KD 120, Chemical Research Division, OntarioHydro, 800 Kipling Avenue, Toronto, Ontario M8Z5S4, Canada. ³Current address: Department of Botany, Washington State University,Pullman, Washington 99164-4230, U.S.A. (Received March 13, 1986; Accepted September 18, 1986)     

Ethylene and 1 -Aminocyclopropane-1-Carboxylic Acid Production in flacca, a Wilty Mutant of Tomato, Subjected to Water Deficiency and Pre-treatment with Abscisic Acid

Neill, S. J., McGaw, B. A. and Horgan, R. 1986. Ethylene and1-aminocyclopropane-l-carboxylic acid production in flacca,a wilty mutant of tomato, subjected to water deficiency andpretreatment with abscisic acid —J. exp. Bot. 37: 535–541. Plants of Lycoperstcon esculentum Mill. cv. Ailsa Craig wildtype and flacca (flc) were sprayed daily with H²O or 2?10^–2mol m^–3 abscisic acid (ABA). ABA treatment effected apartial phenotypic reversion of flc shoots; leaf areas wereincreased and transpiration rates decreased. Leaf expansionof wild type shoots was inhibited by ABA. Indoleacetic acid (IAA), ABA and l-aminocyclopropane-l-carboxylicacid (ACC) concentrations were determined by combined gas chromatography-massspectrometry using deuterium-labelled internal standards ABAtreatment for 30 d resulted in greatly elevated internal ABAlevels, increasing from 1?0 to 4?3 and from 0?45 to 4?9 nmolg^–1 fr. wt. in wild type and flc leaves respectively.Endogenous IAA and ACC concentrations were much lower than thoseof ABA. IAA content ranged from 0?05 to 0?1 nmol g^–1 andACC content from 0?07 to 0?24 nmol g^–1 Ethylene emanationrates were similar for wild type and flc shoots. Wilting of detached leaves induced a substantial increase inethylene and ACC accumulation in all plants, regardless of treatmentor type. Ethylene and ACC levels were no greater in flc leavescompared to the wild type. ABA pretreatment did not preventthe wilting-induced increase in ACC and ethylene synthesis. Key words: ABA, ACC, ethylene, wilting, wilty mutants     

Differences in Zinc Efficiency among and within Diploid, Tetraploid and Hexaploid Wheats

Greenhouse experiments were carried out with six diploid, ninetetraploid and seven hexaploid wheats, including wild and primitivegenotypes, to study the influence of varied zinc (Zn) supplyon the severity of Zn deficiency symptoms, shoot dry matterproduction and shoot Zn concentrations. In addition to wildand primitive genotypes, one modern tetraploid cultivar withhigh sensitivity to Zn deficiency and two modern hexaploid cultivars,one highly sensitive to and one resistant to Zn deficiency,were included for comparison. Plants were grown for 44 d ina severely Zn-deficient calcareous soil, with (+Zn; 5 mg Znkg^-1soil) and without (-Zn) Zn fertilization. Visible Zn deficiencysymptoms, including whitish-brown necrotic patches on leaf blades,appeared very rapidly and severely in all tetraploid wheat genotypes.Compared with tetraploid wheats, diploid and hexaploid wheatswere less sensitive to Zn deficiency. With additional Zn, shootdry matter production was higher in tetraploid than diploidand hexaploid wheats. However, under Zn-deficient conditionstetraploid wheats had the lowest shoot dry matter production,indicating the very high sensitivity of tetraploid wheats toZn deficiency. Consequently, Zn efficiency expressed as theratio of shoot dry matter produced under Zn deficiency to Znfertilization, was much lower in tetraploid wheats than in diploidand hexaploid wheats. On average, Zn efficiency ratios were36% for tetraploid, 60% for diploid and 64% for hexaploid wheats.Differences in Zn efficiency among and within diploid, tetraploidand hexaploid wheats were positively related to the amount ofZn per shoot of the genotypes, but not to the amount of Zn perunit dry weight of shoots or seeds used in the experiments.The seeds of the accessions of tetraploid wild wheats containedup to 120 mg Zn kg^-1, but the resulting plants showed very highsensitivity to Zn deficiency. By contrast, hexaploid wheatsand primitive diploid wheats with much lower Zn concentrationsin seeds had higher Zn efficiencies. It is suggested that notonly enhanced Zn uptake capacity but also enhanced internalZn utilization capacity of genotypes play important roles indifferential expression of Zn efficiency. The results of thisstudy also suggest the importance of the A and D genomes asthe possible source of genes determining Zn efficiency in wheat.Copyright 1999 Annals of Botany Company Seeds, Triticum aestivum, Triticum monococcum, Triticum turgidum, zinc concentrations, zinc deficiency, zinc efficiency.     

CHANGES IN SUBCELLULAR COMPONENTS OF YEAST AS RELATED TO NUCLEOTIDE RELEASE FROM THE CELLS INCUBATED IN CITRATE BUFFER

It was found that when intact cells of a yeast, Saccharomycescerevisiae ‘Yebisu’, were incubated in 0.08 M citratebuffer (pH 6.0) containing 2 per cent glucose, nucleotides werereleased in the medium. In this connection, experiments havebeen carried out to elucidate biochemical changes in subcellularstructure of such cells. Microscopic observation showed that the longer the durationof incubation of the cells in the citrate buffer, the more markedbecomes the granulous appearance of the cytoplasm. Among various subcellular fractions of freshly disrupted cells,the highest content in nucleic acid was found in the cell membranefraction and in the small granule fraction. The nucleic acidcontent in the former fraction decreased markedly, even aftera short period of incubation with citrate, accompanied by anabundant release of nucleotides. In contrast, the nucleic acidcontent in the small granule fraction scarcely changed. Continuedincubation with citrate, however, caused a decrease of nucleicacid content also in this fraction. In this case, also the extracellularrelease of amino acids increased and a partial loss of viabilityof the cells was observed. Ultracentrifugal analysis showedthat the sedimentation pattern of the small granule fraction,consisting of an 80 S (major) and a 40 S (minor) component,did not change on incubation with citrate. ¹Present address. Department of Biochemistry, School of Medicine,Tohoku University, Sendai. (Received May 18, 1962; )     

Purification and Characterization of Phosphoenolpyruvate Carboxylase of Photomixotrophically Cultured Green Tobacco Cells

Phosphoenolpyruvate (PEP) carboxylase (PEPCase, EC 4.1.1.31 [EC] )was purified to apparent electrophoretic homogeneity from photomixotrophicallycultured tobacco cells by ammonium sulfate fractionation, DEAE-Sephacel-,hydroxylapatite-, Phenyl-Sepharose CL-4B-, and Sepharose CL-6B-chromatography,and fast protein liquid chromatography on Mono Q. The purifiedenzyme had a specific activity of 32 units per mg protein, andits purity was determined by denaturing polyacrylamide gel electrophoresis.The native enzyme, with a molecular weight of about 440,000,was a tetramer of four identical subunits and showed maximumactivity at pH 8.5–9.0. Non-denaturing isoelectric focusingshowed a single band at pl 5.4. Substrate-saturation kineticsof the purified enzyme for PEP, bicarbonate, and Mg^2$ were typicalMichaelis-Menten type, with K_m-values of 60, 200, and 80µM,respectively. Most effectors which are known to influence theactivity of C₄- or bacterial PEPCase had only small effectson the activity of the purified enzyme at optimum pH, whilesome inhibitory effects by organic acids (malate, citrate andoxaloacetate) and.an activating effect by glucose-6-phosphatewere observed at a suboptimal pH of 7.5. (Received September 30, 1987; Accepted December 14, 1987)     

The Distribution of Zinc-65 in Agrostis tenuis sibth. and A. stolonifera L. Tissues4

The distribution of ⁶⁵Zn in zinc-tolerant and copper-tolerantplants of Agrostis spp. from toxic mine-tailings in Enflandand Wales was compared with zinc distribution in non-tolerantplants. Isotope was applied in culture solution in which theplants were growing. No differences could be demonstrated betweenthe plants were growing. No differences could be demonstratedbetween the plants by whole-plant radioautography, or by zincanalyses of the tops. Root/shoot ratios calculated from specificactivity values varied with population, the non-tolerant plantshaving the lowest and the zinc-tolerant plants the highest ratio.After solvent (80 per cent ethanol and water) extractions, theroot residue of zinc-tolerant plants contained a higher percentageof ⁶⁵Zn than that of non-tolerant plants. Chemical fractionationof the roots revealed that the main high difference was thatthe amount of ⁶⁵Zn in the pectate extract of the cell wall washigh in zinc-tolerant plants and low in non-tolerant plants.The ⁶⁵Zn distribution in the copper-tolerant plants was similarto that in the non-tolerant plants, indicating that the tolerancemechanisms for the elements are different. Soluble protein andRNA preparations were made but they contained low levels of⁶⁵Zn. An exception was the relatively high value for RNA fromzinc-tolerant A. stolonifera shoots. An anionic complex of ⁶⁵Znin the soluble fraction was investigated. This complex accountedfor most of the radioactivity in A. tennis extracts of shootsbut the concentration of the complex was low in A. stoloniferashoots, and in root extracts of all plants examined.     

Dark Metabolism of CO2 during Fibre Elongation of Two Cottons Differing in Fibre Lengths

A study has been made of the dark metabolism of CO₂ by elongatingfibres of Gossypium arboreum L. cv. LD 133 (a short staple type)and Gossypium hirsutum L. cv. LH 372 (a long staple type) atdifferent fibre ages. In both cultivars, phosphoenolpyruvatecarboxylasc, glutamate-oxalacetate transaminase and malate dehydrogenaseshow elevated activities during the period of rapid fibre growthand lowered activity with ageing. Malic enzyme activity increasesas extension growth levels off. Levels of K⁺ and malate riseduring rapid extension growth and fall as the rate of elongationdecreases. The results indicate that malate may act as an osmoticumand a counterion for K⁺ accumulation during rapid expansionof the fibres. Amounts of enzymes, K⁺ and malate are higherin the fibres of the long staple cultivar than the short staple.During the period of active elongation, K⁺/malate ratio is higherin the short staple cultivar. Key words: Gossypium hirsutum, CO₂ metabolism, Fibre extension     

Short-term Products of 14C-acetate Assimilation by Chlorella pyrenoidosa in the Light

The kinetics of ¹⁴C-2-acetate assimilation by Chlorella pyrenoidosain the light were examined. Under aerobic conditions the primaryproduct of acetate assimilation was succinic acid which, afterten seconds, contained over 60 per cent of the ¹⁴C incorporatedby the cells. The percentage of the total ¹⁴C in succinate fellwith time, while that in citrate and glutamate increased. After1800 sec over 60 per cent of ¹⁴C was present in two compounds,glutamic acid and an unknown compound (X). Glucose-6-phosphate,fructose-6-phosphate, phosphoglyceric acid and phosphoenolpyruvicacid became labelled after 60 sec but together never containedmore than one per cent of the total ¹⁴C incorporated. Underanaerobic conditions succinate was still the primary productof acetate assimilation, and the absence of carbon dioxide resultedin a decrease in ¹⁴C incorporation into compound X. The patternof acetate assimilation in acetate grown and acetate adaptedChlorella was very similar to that in photo-autotrophicallygrown Chlorella. In the presence of 10^–6M DCMU, succinicacid was the primary product of acetate assimilation, but therewas an early Incorporation of ¹⁴C into glutamate, aspartate,and malate. 4 x10^–3M MFA did not effect the early incorporationof ¹⁴C into succinic acid, but resulted in accumulation of ¹⁴Cin citrate and a decreased amount in glutamate and in compound X.     

14CO2 Pulse-Chase Labelling in Clusia minor L.

Conditions and maintenance of growth were chosen so that plantsof Clusia minor L. were obtained which showed the C₃- and CAM-modes of CO₂-exchange, respectively. C. minor is known to accumulateconsiderable amounts of citric acid in addition to malic acidduring the dark-phase of CAM. ¹⁴CO₂-pulse-chase experiments were performed with these plants.Patterns of labelling during the pulse and redistribution oflabel during the chase in the C₃-mode were as expected for C₃-photosynthesis.Pulse-labelling in the CAM-mode during the last hour of thelight period, during the first part of the dark period and duringthe last hour of the dark period always led to an almost exclusiveincorporation of label into malate. Redistribution of labelfrom malate after the pulse at the end of the dark period duringthe chase in the subsequent light period followed the patternexpected for light-dependent reassimilation of CO₂ remobilizedfrom malate in CAM during the light period. During the chasesin the dark period, label was transferred from ^l4C-malate tocitrate. This suggests that during accumulation of citric acidin the dark period of CAM in C. minor, citrate is synthesizedin the mitochondria from malate or oxaloacetate after formationof malate via phosphoenolpyruvate carboxylase. The experiment also showed that no labelled compounds are exportedfrom leaves in the CAM-mode during the dark period. In plantsof the C₃-mode the roots proved to be strong sinks. Key words: Clusia minor, labelling, pulse-chase, ¹⁴CO₂     

Cloning of the Gene Encoding a Protochlorophyllide Reductase: the Physiological Significance of the Co-Existence of Light-Dependent and -Independent Protochlorophyllide Reduction Systems in the Cyanobacterium Plectonema boryanum

Cyanobacteria have two protochlorophyllide (Pchlide) reductasescatalyzing the conversion of Pchlide to chloro-phyllide, a keystep in the biosynthetic pathway of chlorophylls (Chls); a light-dependent(LPOR) and a light-independent (DPOR) reductase. We found anopen reading frame (ORF322) in a 2,131-bp EcoRI fragment fromthe genomic DNA of the cyanobacterium Plectonema boryanum. Becausethe deduced amino acid sequence showed a high similarity tothose of various plant LPORs and the LPOR activity was detectedin the soluble fraction of Esche-richia coli cells over-expressingthe ORF322 protein, ORF322 was defined as the por gene encodingLPOR in P. boryanum. A por-disrupted mutant, YFP12, was isolatedby targeted mutagenesiss to investigate the physiological importanceof LPOR. YFP12 grew as well as wild type under low light conditions(10-25 µE m^–2 S^–1). However, its growth wassignificantly retarded as a result of a significant decreasein its Chl content under higher light conditions (85-130 µEm^–2 s^–1). Furthermore, YFP12 stopped growing andsuffered from photobleaching under the highest light intensity(170 µE m^–2 s^–1). In contrast, a chlL-dis-rupted(DPOR-less) mutant YFC2 grew as well as wild type irrespectiveof light intensity. From these phenotypic characteristics, weconcluded that, although both LPOR and DPOR contribute to Chlsynthesis in the cells growing in the light, the extent of thecontribution by LPOR increases with increasing light intensity;without it, the cells are unable to grow under light intensitiesof more than 130 µ Em^–2s-^–. (Received September 26, 1997; Accepted November 21, 1997)     

Properties of Leaf NAD-Malic Enzyme from the Inducible Crassulacean Acid Metabolism Species Mesembryanthemum crystallinum

NAD-malic enzyme (NAD-ME) functions to decarboxylate malatein the light in leaves of certain species displaying Crassulaceanacid metabolism (CAM). The properties of NAD-ME in desaltedextracts from the inducible CAM species, Mesembryanthemum crystallinumwere examined. The shapes of the malate saturation curve andthe activity versus pH curve at 10 mM malate were dependenton the presence of the activator CoA. The malate saturationcurve was sigmoidal in the absence of an activator and hyperbolicin the presence of CoA. The pH optimum with 10mM malate andMn²⁺ as cofactor was as low as 6.5 without an activator, andincreased to 7.2 in the presence of CoA. Fumarate activationwas synergistic with CoA above pH 7.2. The enzyme displayedhysteretic behavior under suboptimal assay conditions. Rapid extraction and desalting of the enzyme (<1.5 mim) followedimmediately by assay did not reveal any difference in the propertiesof the enzyme on a day/night basis. It is proposed that diurnalregulation of the enzyme in vivo is mediated by pH and malatelevel without a change in the oligomeric form of the enzyme.The molecular weight of the enzyme was approximately 350,000at pH 6.5 or 7.8. The enzyme obtained from M. crystallinum inthe C₃ mode was very similar to the CAM enzyme except that itdisplayed a lower V_max. ³ Current address: MSU-DOE Plant Research Lab, Michigan StateUniversity, E. Lansing, Michigan, U.S.A. 48824. (Received October 2, 1984; Accepted December 20, 1984)     

Induction of Low Molecular Weight Cadmium-Binding Compound in Soybean Roots

Blakeley, S. D., Robaglia, C, Brzezinski, R. and Thirion, J-P.1986. Induction oflow molecular weight cadmium-binding compoundin soybean roots.—J. exp. Bot. 37: 956–964. A Cd-binding compound has been identified in roots of Cd-treatedsoybean plants (Glycine max L.). Induction of synthesis of thiscompound after treatment with CdCl₂ was monitored by labellingwith either ¹⁰⁹Cd in vitro or with ³⁵S-cysteine or ³⁵S-methioninein vivo. The apparent molecular weight as determined by gelfiltration was about 14000 daltons. However, after carboxymethylationand electrophoresis under denaturing conditions its molecularweight was less than 3400 daltons. Simultaneous labelling with¹⁰⁹ and ³⁵S-cysteine demonstrated that the compound containscysteine residues. Gel electrophoresis of ³⁵S-cysteine or ³⁵S-methioninelabelled protein samples showed the compound to be rich in cysteinebut not in methionine, and that it was induced about 60-foldby 1?0 mol m^–3 CdCl₂ after 7 d. Key words: Soybean, cadmium, induction, root     

ERRATA

Page 678, line 3, for [4-¹⁴C] read [I-¹⁴C] Page 678, line 4, for [I-¹⁴C] read [4-¹⁴C] Page 679, line 17, for C-I of malate read C-4 of malate Page 679, line 18, for C-4 of malate read C-I of malate     

Amounts of Nuclear DNA and Internal Morphology of Gibberellin- and Abscisic Acid-deficient Tomato (Lycopersicon esculentum Mill.) Seeds During Maturation, Imbibition and Germination

Using X-ray photography and flow cytometry, the internal morphologyand DNA replication activity of wild type (wt), GA- (gib-1 )and ABA-deficient (sit^w ) tomato (Lycopersicon esculentum Mill.cv. Moneymaker) mutant seeds were studied. During seed formation,from 30 to 45 d after pollination (DAP) the endosperm becomessolid and the seed starts to gain desiccation tolerance. Atthis time significant changes occur in the amounts of DNA inradicle tip cells. At 30 DAP, radicle tip cells of the threegenotypes manifest about 60% of 2C, 30% of 4C and 10% of 8Camounts of DNA. Upon maturation (45 DAP onwards), most cellsin the seeds of the three genotypes arrest in the G₁phase ofthe cell-cycle with 2C amounts of DNA. However, a relativelyhigh proportion of cells with 4C amounts of DNA was detectedin the radicle tip cells ofsit^w compared with wild type andgib-1. At the well-matured stage (60 DAP), there were about 2% ofseeds with free space in wild type andgib-1 , and about 13%insit^w . At the over-matured stage (75 DAP), even more seedswith free space were found insit^w , whereas no increase in theproportion of the seeds with free space was detected in theother two genotypes. In -1.0 MPa PEG-6000 with or without 10µM GA₄₊₇, no germination occurred in well-matured wildtype andgib-1 seeds, whether or not they were dried after harvest.However,sit^w seeds were able to germinate both in over-maturefruit and in -1.0 MPa PEG-6000. Priming of dried seeds in -1.0MPa PEG induced a large amount of free space in almost all seedsof the three genotypes, and nuclear DNA synthesis in the radicletip cells of wild type andsit^w seeds. However, PEG priming offresh (non-dried) seeds had no effect on the amount of freespace and 2C/4C DNA ratios in wild type orgib-1 seeds, but didinduce free space in about 20–25% ofsit^w seeds and provoked4C signals insit^w seeds. Removal of the endosperm and testaopposite the radicle tip of seeds resulted in root protrusion,the induction of free space and an increase of 4C DNA signalsin the three genotypes. It is concluded that ABA is crucialfor the efficient arrest of tomato embryo radicle tip cellsin G₁phase upon maturation, whereas GAs play an important rolein re-initiating 4C DNA levels upon germination. Dormancy; flow cytometry; free space; Lycopersicon esculentum ; maturation; priming; seed; tomato     

Guard Cell Metabolism in Epidermis of Commelina communis L. During Stomatal Opening and Closing

The rates of CO² incorporation into the epidermis of C. communiswere linear and were similar during the completion of opening(2 h) and closing (1 h) movements of stomata. The kinetics of¹⁴C turnover between metabolites and the rates of ‘leakage’of metabolites were determined for opening and closing movements.When stomata were opening there was a slow turnover of ¹⁴C frommalate chiefly into sugars. Upon stomatal closure ¹⁴C was initiallymainly in sugars, malate, and sugar phosphates. Thereafter,there was a slight loss of label from sugar phosphates witha corresponding increase in malate. Starch became labelled duringopening and closing movements. Rates of incorporation of CO₂found in the ‘leakage’ fraction were greatest whenstomata were opening. Of the labelled compounds Most‘from the tissue, malate was the most highly labelled whetherstomata were opening or closing. Although interpretation of the turnover patterns is difficultwithout knowledge of pool sizes for the metabolites it is suggestedthat a pool of sugars exists within the guard cells, which havefairly direct and reversible access to carbon from starch andmalate. The implications of loss of malate from guard cellsduring stomatal opening and closing are discussed.