Alteration of Soybean Seedling Development in Darkness and Light by the Stay-green Mutation cytG and Gd1d2

The stay-green mutations cytG and Gd₁d₂ prevent the normal yellowingduring senescence of soybean (Glycine max) leaves and cotyledons.Because light plays such an important role in regulating morphogenesisand it promotes the formation of chlorophyll (Chl), we determinedthe effect of cytG and Gd₁d₂ (in a cv. Clark background) onthe development and some light responses of seedlings. AlthoughcytG and Gd₁d₂ seeds, particularly the cotyledons, are greenwhen mature, 44 and 71 % respectively of this Chl broke downwhen the seeds were germinated in darkness. Chlorophyllidesand phaeophytins were not present in the seeds in significantamounts. cytG and Gd₁d₂ as well as wild type (cv. Clark) seedlingsdeveloped a full etiolation syndrome (morphology and lack ofChl) in darkness. Light induced rapid Chl accumulation in thedark-grown seedlings with no apparent difference among the threeisolines. A short (8 h) exposure to light induced some Chl inthe cotyledons of dark-grown plants, and 22 h of light producedfour times more. Following return to darkness, the 8-h groupshowed very little breakdown over the next 12 d. After the 22-hgroup was returned to darkness, the wild-type lost Chl steadily,but Gd₁d₂ and eventually also cytG inhibited this breakdown.In the 22-h group, the Chl a/b ratio decreased in wild typeand cytG indicating preferential breakdown of Chl a relativeto Chl b; however, Gd₁d₂ prevented this change. cytG and Gd₁d₂seem to act preferentially on Chl breakdown rather than synthesis.Copyright1995, 1999 Academic Press Glycine max, soybean, chlorophyll, chlorophyll a/b ratio, cotyledons, etiolation, cytG, Gd₁d₂, mutations, senescence     

CO2-Fixation, Glycolate Formation, and Enzyme Activities of Photorespiration and Photosynthesis during Greening and Senescence of Sunflower Cotyledons

Time-courses of ¹⁴CO₂-fixation and of enzyme activities involvedin photorespiration and photosynthesis were determined duringthe life span of cotyledons from sunflower seedlings (Helianthusannuus L.). Glycolate formation in vivo was estimated from theresults of combined labelling and inhibitor experiments. NADPH-glyceraldehyde-3-phosphatedehydrogenase, NADPH-glyoxylate reductase and chlorophyll werewell correlated with the time-course of ¹⁴CO₂-fixation (photosynthesis).There was, however, a considerable discrepancy between the developmentalsequence of photosynthesis and that of both ribulose-l,5-bisphosphatecarboxylase and glycolate oxidase. Furthermore, time-coursesof glycolate oxidase activity in vitro and of glycolate formationin vivo differed significantly. Therefore, the use of glycolateoxidase as a marker for the activity of photorespiration ingreening sunflower cotyledons may be questionable. Results from¹⁴CO₂-labelling experiments with cotyledons treated with theglycolate oxidase inhibitor 2-hydroxy butynoic acid suggestthat glycolate formation relative to CO₂-fixation is reducedin senescent cotyledons. Key words: Development, glycolate oxidase, photorespiration, ribulose-l,5-bisphosphate carboxylase, oxygenase     

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; )     

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     

Alanine aminotransferase from Cucurbita moschata cotyledons

Alanine aminotransferase increased in pumpkin cotyledons duringgermination with the greatest increase occurring in green cotyledons.The enzyme was found in the soluble fraction and was inhibitedby NH₂OH and p-chloromercuribenzoate. Pyridoxal phosphate andglutathione or dithiothreitol overcame die respective inhibition.Dialysis of the enzyme reduced enzyme activity but the activitywas restored by the addition of pyridoxal phosphate. Alanineaminotransferase was proposed to play a major role in the synthesisof the alanine which occurs in pumpkin cotyledons during germination. (Received September 17, 1975; )     

Metabolism and Translocation of Gibberellins in Seedlings of Pharbitis nil. (I) Effect of Photoperiod on Stem Elongation and Endogenous Gibberellins in Cotyledons and Their Phloem Exudates

Gibberellin A₁, (GA₁), GA₁₉, and GA₂₀ in phloem exudates andcotyledons of seedlings of Pharbitis nil cv. Violet, grown underdifferent photoperiodic conditions, were qualitatively and semi-quantitativelyanalyzed by a combination of high performance-liquid chromatography(HPLC) and radioimmunoassays (RIA). The levels of GA₁₉ and GA₂₀were higher in cotyledons from plants grown under dark treatment(DT) conditons of 16 h-light/8 h-dark for 6 days followed by8 h-light/16 h-dark for 3 days than in those grown under continuouslight (CL) for 9 days. This relationship was also observed forthe GAs in phloem exudates, although the levels were much lowerthan in the cotyledons. When GAs were applied to the cotyledons,elongation of the epicotyl was promoted more by GA₂₀ than byGA₁ or GA₁₉, especially under the CL treatment. The relativeeffect of GA₁ and GA₂₀ on the epicotyl elongation was reversedwhen these GAs were applied to epicotyls pre-treated with prohexadione,an inhibitor of 2-oxoglutarate-dependent dioxygenases. ³Present address: Frontier Research Program, The Institute ofPhysical and Chemical Research (RIKEN), 2-1 Hirosawa, Wakoshi,Saitama, 351-01 Japan ⁴Present address: Laboratory of Horticulture, Faculty of Agriculture,Nagoya University, Nagoya, 464-01 Japan     

Photoactivation of Oxygen-Evolving Enzyme in Dark-Grown Pine Cotyledons: Relationship between Assembly of Photosystem II Proteins and Integration of Manganese and Calcium

Dark-grown cotyledons of pine (Pinus thunbergit) did not exhibitO₂ evolution, but this capability was rapidly activated by illuminationfor a short period (photoactivation). To examine the biochemicalchanges which accompany the process of photoactivation in gymnosperms,a method enabling the preparation of highly active O₂-evolvingphotosystem II (PS II) membranes was applied to light-grown,dark-grown, and photoactivated cotyledons. PS II membranes preparedfrom light-grown cotyledons exhibited high O₂-evolving activity,and contained all the intrinsic proteins as well as the threeextrinsic proteins (32, 23 and 17 kDa) associated with PS II.These membranes were also found to contain 4.4 Mn and 0.83 Ca/PSII reaction center. PS II membranes from dark-grown cotyledonscontained all the intrinsic proteins, but preserved only 32kDa extrinsic protein, and zero Mn and 0.85 Ca/PS II reactioncenter. The two extrinsic proteins (23 and 17 kDa) absent inthe PS II membranes from dark-grown cotyledons were, however,present as mature forms in whole thylakoid membranes from thecorresponding sample. The PS II membranes isolated from photoactivatedcotyledons showed a high activity of O₂ evolution and retainedthe three extrinsic proteins, 5.3 Mn and 1.1 Ca/PS II reactioncenter, respectively. The results indicated that Mn and thetwo extrinsic proteins were tightly integrated in the O₂-evolvingapparatusduring the process of photoactivation but integration of Capreceded the integration of Mn by photoactivation. (Received December 9, 1991; Accepted February 1, 1992)     

Influence of Axis Removal on Amino-, Carboxy- and Endopeptidase Activities in Cotyledons of Germinating Vigna mungo Seeds

Endopeptidase (azocaseolytic enzyme) and carboxypeptidase activitiesin cotyledons of germinating Vigna mungo seeds increased until3 days after the onset of imbibition and decreased thereafter.In detached and incubated cotyledons, the endopeptidase activityincreased only a little while the carboxypeptidase activitycontinued increasing even after 3 days of incubation. The activitiesof leucine-aminopeptidase and alanine-aminopeptidase, exceptfor that of one leucine-aminopeptidase isoenzyme relativelyabundantly present in unimbibed dry cotyledons, increased slightlyon the first day and declined during germination. In detachedcotyledons, the activities maintained their initial levels throughoutthe incubation period. When cotyledons were detached from germinatingseedlings on days 2 and 4 then incubated, the endopeptidaseactivity started to decrease just after removal of the axisbut the carboxypeptidase activity increased more markedly thanwhen the axis remained attached. Exogenously supplied GA₃, kinetin,IAA, or their combinations, showed no significant effect onthe developmental patterns of the endopeptidase and carboxypeptidaseactivities in cotyledons. These results are discussed in relationto the role of the axis in controlling peptidase formation incotyledons of germinating V. mungo seeds. (Received November 18, 1983; Accepted February 28, 1984)     

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     

Photosynthesis and Light Activation of Ribulose 1, 5-bisphosphate Carboxylase in the Presence of Starch

Owing to a typographical error three equations were omittedfrom page 1294. The correct paragraphs are set out below. The component K₁ corrected for the difference in temperaturebetween the enzyme assay and the leaf and was calculated accordingto the Arrhemus equation. where v10 and v18 are the reaction velocities of carboxylationat 10?C and 18?C, respectively and A is the activation energy(A = 90 kJ mol^–1, as determined for purified wheat RuBPCOby M?chler, Keys and Cornelius, 1980) The components K2 corrected for the difference in CO₂ partialpressure between enzyme assay and leaf and for competitive inhibitionof carboxylation by O₂ and was calculated according to the modifiedMichaelis Menten equation where v_c, is the carboxylation velocity under leaf conditions,V_c. is the maximum carboxylation velocity as determined in theenzyme assay, K_c, and K_o are the Michaelis constants for carboxylationand oxygenation, respectively (K_o = 159 Pa CO₂. K_o = 35.3 kPaO₂, as interpolated for 18?C from spinach data as determinedby Jordan and Ogren, 1984), O is oxygen partial pressure inair and C₁ is intercellular CO₂ partial pressure in leaves (C₁= 29.1 ? 0.8 Pa (? s c , n = 15)) The component K3 corrected for the decrease in CO₂ fixationin leaves due to photorespiration and was calculated accordingto equation 3 Equation 3 is denved from the equation for the substrate specificityof RuBPCO, S= v_c/v_oC (Laing, Ogren, and Hageman, 1974), andfrom the equation for the stoichiometry of photorespiratoryCO₂ release, F=v_c–1/2 v_o, where v_c, and v_c are reactionvelocities of carboxylation and oxygenation, O and C are partialpressures of 02 and intercellular CO₂, F is net photosynthesisand S is the substrate specificity of RuBPCO (S= 3061 Pa/Pa,as interpolated for 18?C from spinach data as determined byJordan and Ogren, 1984)     

Photosynthesis in Panicum milioides, a Species with Reduced Photorespiration

The capacity for C₄ photosynthesis in Panicum milioides, a specieshaving reduced levels of photorespiration, was investigatedby examining the activity of certain key enzymes of the C₄ pathwayand by pulse-chase experiments with ¹⁴CO₂. The ATP$P₁ dependentactivity of pyruvate,P₁ dikinase in the species was extremelylow (0.14–0.18 µmol mg chlorophyll^–1 min^–1).Low activity of the enzyme was also found in Panicum decipiensand Panicum hians (related species with reduced photorespiration)and in Panicum laxum (a C₃ species). The antibody to pyruvate,P₁dikinase caused about 70% inhibition of the ATP$P₁ dependentactivity of the enzyme in P. milioides. The activity of NAD-malicenzyme and NADP-malic enzyme in ^P. ^milioides was equally low(approximately 0.1–0.2 µmol mg chlorophyll^–1min^–1) and similar to the activity in P. decipiens, P.hians and P. laxum. Photosynthetic pulse-chase experiments underatmospheric conditions showed a typical C₃-like pattern of carbonassimilation including the labelling of glycine and serine asexpected during photorespiration. During the pulse with ¹⁴CO₂only about 1% of the labelled products appeared in malate and2–3% in aspartate. During a chase in atmospheric levelsof CO₂ for up to 6 min there was a slight increase in labellingin the C₄ acids. The amount of label in carbon 4 of aspartatedid not change during the chase, indicating little or no turnoverof the C₄ acid via decarboxylation. The results indicate thatunder atmospheric conditions P. milioides assimilates carbondirectly through the C₃ pathway. Photorespiration as indicatedby the CO₂ compensation point may be repressed in the speciesby a more efficient recycling of photorespired CO₂. (Received June 8, 1982; Accepted July 22, 1982)     

A Novel Isourazole Herbicide, Fluthiacet-Methyl, is a Potent Inhibitor of Protoporphyrinogen Oxidase after Isomerization by Glutathione S-Transferase

A novel isourazole herbicide, fluthiacet-methyl (methyl [[2-chloro-4-fluoro-5-[(5,6,7,8-tetrahydro-3-oxo-lH,3H-[l,3,4]thiadiazolo[3,4-a]pyridazin-l-ylidene)amino]phenyrjthio]acetate;experimental code name, KIH-9201) promoted the leakage of electrolytesfrom cotyledons of velvetleaf (Abtilon theophtasti Medic) andcotton (Gossypium hirsutum L.) plants that are sensitive tothis compound. It induced the accumulation of protoporphyrinIX in cotyledons of cotton and inhibited Chl biosynthesis incotyledons of velvetleaf and cotton at low concentrations (I₅₀values, 10–12 nM). Fluthiacet-methyl was converted toits urazole by glutathione S-transferase that had been partiallypurified from velvetleaf. The urazole inhibited protoporphyrinogenoxidase (Protox, EC 1.3.3.4 [EC] ) from some plants, including velvetleaf,at low concentrations (I₅₀ values, 5.1–11 nM), whereasfluthiacet-methyl was not as potent. The effects in vivo (electrolyteleakage and inhibition of Chi biosynthesis) of fluthiacet-methylwere correlated with the inhibition of Protox activity by theurazole and not with the action of fluthiacet-methyl itself.From these results, it is concluded that fluthiacet-methyl inhibitsProtox activity after conversion to the corresponding urazoleby glutathione S-transferase. It is in this way that fluthiacet-methylexerts its effect as a light-dependent peroxidizing herbicide. (Received November 1, 1994; Accepted March 6, 1995)     

The Greenhouse Effect: Acclimation of Tomato Plants Growing in High CO2, Photosynthesis and Ribulose-1, 5-Bisphosphate Carboxylase Protein

Tomato plants were grown in solution culture in a controlledenvironment at 20 ?C with a 12 h photoperiod of 400 µmolquanta m^–2 s^–1 PAR with either normal ambient CO₂,approximately 340 vpm, or with 1000 vpm CO₂. The short- andlong-term effects of CO₂ enrichment on photosynthesis were determinedtogether with the levels of ribulose-1, 5-bisphosphate carboxylase(RuBPco) E.C. 4.1.1.39 [EC] protein and activity throughout leafdevelopment of the unshaded 5th leaf above the cotyledons. Thehigh CO₂ concentration during growth did not appreciably affectthe rate of leaf expansion or final leaf area but did increasethe fresh weight per unit area of leaf. With short-term CO₂enrichment, i.e. only during the photosynthesis measurements,the light-saturated photosynthetic rate (P_max) of young leavesdid not increase while those reaching full expansion more thandoubled their net rate of CO₂ fixation. However, with longerterm CO₂ enrichment, i.e. growing the crop in high CO₂, theplants did not maintain this photosynthetic gain. While theCO₂ concentration during growth did not affect the peak in P_maxmeasured in 300 vpm CO₂ or P_max in 1000 vpm CO₂, RuBPco proteinor its activity, the subsequent ontogenetic decline in theseparameters was greatly accelerated by the high CO₂ treatment.Compared with plants grown in normal ambient CO₂ the high CO₂grown leaves, when almost fully expanded, contained only approximatelyhalf as much RuBPco protein and P_max in 300 vpm CO₂ and P_maxin1000 vpm CO₂ were similarly reduced. The loss of RuBPco proteinmay be a major factor associated with the accelerated fall inP_max since it was close to that predicted from the amount andkinetics of RuBPco assuming RuBP saturation. In the oldest leavesexamined grown in high CO₂ additional factors may be limitingphotosynthesis since RuBPco kinetics marginally overestimatedP_max in 300 vpm CO₂ and the initial slope of photosynthesisin response to intercellular CO₂ was also less than expectedfrom the extractable RuBPco. Key words: Lycopersicon esculentum (Mill.) cv. Findon Cross, CO₂ enrichment, acclimation to high CO₂, photosynthesis, RuBPco protein and activity     

Effect of Sink-Source Manipulations on the Photosynthetic Rate and Carbohydrate Content of Cucumber Cotyledons

Mayoral, M. L., Plaut, Z. and Reinhold, L. 1985. Effect of sink-sourcemanipulations on the photosynthetic rate and carbohydrate contentof cucumber cotyledons.-J. exp. Bot. 36 1551–1558. The photosynthetic rate of cucumber cotyledons (Cucumis sativuscv. Dahla) reached a maximum value of 12 mg dm^–2 h^–1,10 d after emergence. In 12-d-old seedlings removal of one cotyledondoubled the CO₂ fixation rate of the other, as observed 3 dafter treatment. When the primary leaf was removed, the photosyntheticrate of the cotyledons was decreased by 33%. At this stage ofgrowth elimination of the roots as a sink for assimilates bygirdling the hypocotyl affected neither the photosynthetic ratenor the carbohydrate content of the cotyledons. By contrast,in 18-d-old seedlings removal of the first leaf brought abouta 42% increase in the photosynthetic rate of the cotyledons.The simultaneous removal of the first leaf and one cotyledondoubled the rate of CO₂ fixation of the remaining cotyledon.Girdling the hypocotyl lowered the photosynthetic rate of thecotyledons by 73%. In both 12- and 18-d-old seedlings a decreaseor increase in the sink-source ratio was correlated with anincrease or a decrease respectively in the carbohydrate contentof the cotyledons. The stomatal resistance of the cotyledonswas not affected by any of the treatments. The effect of sink-sourcemanipulations on photosynthesis and on the level of carbohydratespresent in the cotyledons was more evident in those seedlingsgrowing under high light intensity (580 µE m^–2 s^–1),than in those exposed to 300 µE m^–2 s^–1 Key words: Sink-source relationship, cotyledons, photosynthesis     

Comparison of carbon dioxide fixation and the fine structure in various assimilatory tissues of Amaranthus retroflexus L

The pattern for primary products of CO₂-fixation and the chloroplaststructure of Amaranthus retrqflexus L., a species which incorporatescarbon dioxide into C₄ dicarboxylic acids as the primary productof photosynthesis, were compared in various chlorophyll containingtissues,i.e., foliage leaves, stems, cotyledons and pale-greencallus induced from stem pith. Despite some morphological differencesin these assimilatory tissues, malate and aspartate were identifiedas the major compounds labelled during a 10 sec fixation of¹⁴CO₂ in all tissues. Whereas, aspartate was the major componentin C₄-dicarboxylic acids formed in foliage leaves, malate predominatedas the primary product in stems, cotyledons and the pale-greencallus. The percentage of ¹⁴C-radioactivity incorporated intoPGA and sugar-P esters increased and ¹⁴C-sucrose was detectedin the prolonged fixation of ¹⁴CO₂ in the light, not only infoliage leaves, but also in stems and cotyledons. ¹ This work was supported by a Grant for Scientific ResearchNo. 58813, from the Ministry of Education, Japan. ² Present address: Institute of Applied Microbiology, Universityof Tokyo, Tokyo, Japan. ³ Present address: Department of Biochemistry, University ofGeorgia, Athens 30601. Georgia, U. S. A. (Received July 10, 1971; )     

Variation in the DNA Content of Glycine Species

Nuclei were isolated from cotyledons of a range of accessionsfrom 14 species of Glycine. These were stained with ethidiumbromide and the relative fluorescence for each genotype wasmeasured by flow cytometry. The DNA content was estimated bycomparison of relative fluorescence with that from nuclei fromseedling leaves of Allium cepa, whose DNA content has been calculatedpreviously by chemical assay. The 4C amounts for diploid Glycineranged from 3.80 to 6.59 pg. Two groups of diploid species appearedfrom the analysis. The first consisted of species with amountsranging from 3.80 to 5.16 pg and included G. canescens (AA),G. argyrea (A₁ A₁), G. clandestina (A²A²), G. microphylla(BB),G. latifolia (B₁B₁), G. tabacina 2n=40 (B²B²), G. tomentella2n=38 (EE) and 2n=40 (DD), G. max and G. soja (GG), G. arenariaand G. latrobeana. A second group had higher DNA contents rangingfrom 5.27 to 6.59 pg, and consisted of G. curvata, G. cyrtoloba(CC), and G. falcata (FF). The polyploid species, G. tabacina2n=80 (AABB, BBB¹B¹), G. tomentella 2n=78 and 2n=80 (AAEE andDDEE, respectively) contained amounts approximating to the sumsof the respective parental diploid species thought to have givenrise to these allotetraploids. Intraspecific variation was detectedin the DNA content of G. canescens. Within the overall distributionof DNA amounts found in A genome species, each genome containeda range of DNA contents specific to that species. This phenomenonwas also detected amongst B genome species.     

Isocitrate Lyase from Germinating Soybean Cotyledons: Purification and Characterization

Ruchti, M. and Widmer, F. 1986. Isocitrate lyase from germinatingsoybean cotyledons: purification and characterization.—J.exp. Bot. 37: 1685–1690. Isocitrate lyase (E.C. 4.1.3.1 [EC] ) was purified from the cotyledonsof 7-d-old soybean seedlings. Three molecular forms were detectedwith pi values of 6·46, 6·25 and 6·0. Themain form (pl = 6·46) had an approximate Mr of 130000,a pH optimum of 8·0, a K_m (isocitrate) close to 2·0mol m^–3 and a molecular activity of 615 min ^–1 at25 °C. The purified enzyme is not a glycoprotein and isheat labile. Key words: Isocitrate lyase, soybean     

Photosynthetic CO2-fixing activity in dark-grown spruce seedlings

Bicarbonate-dependent O₂-evolving activity in dark-grown cotyledonsof Picea abies was measured with an oxygen electrode with differentpreillumination times. The activity showed a slight linear increasewith increasing preillumination time. On the other hand, O₂-evolvingactivity (Hill activity) of chloroplasts prepared from preilluminateddark-grown cotyledons exhibited a characteristic change of asteep rise followed by a gradual increase with increasing preilluminationtime. The results obtained were discussed in connection withthe light activation of the latent, inactive O₂-evolving centerin dark-grown cotyledons. (Received December 8, 1978; )     

Limitations on the Analysis of Acetylene Reduction by Soybean at Low Levels of Acetylene

The analysis of acetylene reduction at low concentrations ofacetylene involves a number of assumptions and both technicaland kinetic complexities. The major difficulty in convertingacetylene reduction rates to apparent N₂ reduction rates isdetermining the K_m for acetylene in the presence of N₂. Thissubstrate competition is dominant over diffusion limitationeffects, but both introduce equivalent deviations in the observedK_m. Because N₂ is a non-linear partial competitive inhibitorof acetylene reduction, correction for its presence is difficult.Two further complications are introduced by the non-linear responseof nitrogenase to acetylene concentration even in the absenceof N₂, and changes in the apparent K_m of acetylene and K₁ ofN₂ as a function of other variables in the enzyme assay. Itis proposed that transient analysis may be used for measurementof diffusion coefficients and calculations of possible diffusionlimitations. It is demonstrated that one proposed model forestimating diffusion limitation (Denison et al., 1983, PlantPhysiology 73, 648–51) confounds substrate competitionwith diffusion limitation. Acetylene reduction, nitrogen fixation, diffusion limitation     

Inhibition of Zeatin-Induced Growth of Cucumber Cotyledons by Sulfite Ions

The inhibitory effects of sulfite ions on zeatin-induced cellexpansion in cotyledons excised from dark-grown seedlings ofcucumber (Cucumis sativus L.) were examined. With 50 µMzeatin the growth rate in white light was about twice that ofthe control. Addition of Na₂SO₃ in the growth medium inhibitedthe zeatin-induced growth of cotyledons. Zeatin-treatment increasedthe osmotic potential in cell sap of cotyledons, while sulfitedecreased it. These treatments had no significant effect onpotassium concentration. Sulfite inhibited the zeatin-inducedincrease in contents of fructose and glucose, but did not affectsucrose content. The relative contents of non-cellulosic constituentsof cell walls fell with the advance of culture. This decreasewas repressed by sulfite, indicating that inhibition of expansiongrowth in cucumber cotyledons by sulfite ions was the resultof alterations in the cell wall structure due to changes inthe cell wall metabolism. (Received June 12, 1984; Accepted October 24, 1984)