Gas Chromatography-Flame Thermionic Detector Analysis of Cytokinins in Etiolated Squash Seedlings using 14C-BA as an Internal Standard

Cytokinin contents in cotyledon, hypocotyl and root of etiolatedsquash (Cucurbita maxima Duch.) seedlings were determined byinstrumental analysis using ¹⁴C-benzyladenine (¹⁴C-BA) as aninternal standard. Crude extracts were purified using insolublepolyvinylpyrrolidone, cellulose-phosphate column and SEP-PAKC18 cartridge, then applied to a Sephadex LH-20 column to separatezeatin riboside (ZR), isopentenyl adenosine, isopentenyl adenine,¹⁴C-BA and a mixture of zeatin (Z) and dihydrozeatin (DHZ).The recovery rate for the cytokinin fractions after LH-20 wascorrected by ¹⁴C-BA. Each cytokinin fraction was further purifiedby HPLC which also separated Z and DHZ in the LH-20 fraction.Before permethylation, ¹⁴C-BA was added to each of the cytokininfractions to correct the methylation rates. Each methylatedcytokinin fraction was again purified by HPLC, then subjectedto gas chromatography with a capillary column and flame thermionicdetector. The detection limit of cytokinins by this system was0.1 ng. cis-ZK was the most abundant cytokinin in all tissues of theetiolated squash seedlings. Active cytokinins such as trans-ZRand trans-Z were mostly found in cotyledons with lesser amountsin the roots. DHZ was most abundant in the cotyledon. All cytokininsisolated by this procedure were confirmed by gas chromatographyselectedion monitoring. (Received December 26, 1986; Accepted June 1, 1987)     

Relation between Sex Expression Types and Cotyledon Etiolation of Cucumber in vitro I. On the Role of Ethylene Evolved from Seedlings

Cucumber seedlings, when cultured in vitro, showed differencesin cotyledon etiolation rates among cultivars with differentgenetic backgrounds for sex expression. The chlorophyll contentin gynoecious cultivars (acr^F/acr^F) decreased rapidly whilethat in monoecious ones (acr⁺/acr⁺) decreased more slowly, andthat in mono-gynoecious ones (acr¹/acr¹) decreased at an intermediaterate. Etiolation was suppressed even in early-etiolating cultivarswhen the flask remained unsealed or endogenously evolved ethylenewas removed. Cotyledon etiolation was enhanced even in late-etiolatingcultivars when ethephon was added to the flask. The rate ofetiolation corresponded to the ethylene concentration in theflask; much more ethylene was detected in early-etiolating cultivarsthan in late-etiolating ones. Ethylene accumulation is one of the important factors involvedin the cotyledon etiolation observed in in vitro cultures. Thedifference in etiolation rates among seedlings with differentgenetic backgrounds for sex expression corresponds to theirability for ethylene evolution, in the order of acr^F>acr¹>acr⁺. (Received January 6, 1981; Accepted March 23, 1981)     

Osmotic Adjustment and Osmotic Constituents in Roots of Mung Bean Seedlings

Osmotic adjustment in roots of mung bean seedlings (Vigna mungo(L.) Hepper) and the effect of cotyledon excision on the osmoticadjustment were investigated. The major osmotic constituentsin roots of intact seedlings were K⁺, Cl^–, free aminoacids and sugars (glucose, fructose and sucrose). All theseintracellular concentrations distinctly increased under osmoticstress and contributed to about 80% of the intracellular osmoticpressure of the root cell sap. Cotyledon excision remarkablysuppressed both the osmotic adjustment and the elongation inroots. However, the effect of cotyledon excision on intracellularK⁺ and Cl^– concentrations in roots was quite small. Twodifferent mechanisms are likely for the osmotic adjustment inroots. One is the K⁺ and Cl^–-dependent osmotic adjustmentwhich is cotyledon-independent, and the other is the osmoticadjustment dependent on the supply of free amino acids and sugarsfrom cotyledons. (Received September 20, 1986; Accepted January 14, 1987)     

Reconstitution and Characterization of H+-Translocating ATPase from the Plasma Membrane of Phaseolus mungo L. Roots

Plasma membrane H⁺-translocating ATPase was partially purifiedfrom mung bean (Phaseolus mungo L.) roots and reconstitutedinto soybean phospholipid (asolectin) liposomes by the n-octylglucosidedilution method. The resulting proteoliposomes were mainly unilamellarvesicles ranging in size from 0.05 to 0.2 µm. The existenceof ATP-drived H⁺-pumping across the proteoliposomes was demonstratedby the quenching of quinacrine fluorescence in the presenceof Mg²⁺. The quenching could be abolished by an uncoupler, FCCP,and an inhibitor of H⁺-translocating ATPase, vanadate. The reconstitutedATPase consisted of three major polypeptides of 105 KDa, 67KDa and 57 KDa. Its pH optimum, divalent cation stimulationand vanadate sensitivity were similar to those of partiallypurified ATPase. However, the specificity toward ATP was muchgreater following reconstitution. Also reconstitution reducedthe degree of inhibition by DCCD. Local anesthetics (e.g. dibucaine)had no effect on H⁺-pumping activity but increased the ATPaseactivity when proteoliposomes were reconstituted in their presence. (Received May 2, 1986; Accepted October 17, 1986)     

The Inhibition of Flowering by Non-Induced Cotyledons of Pharbitis nil

Inhibitory effects on flowering of a non-induced cotyledon havebeen examined in Pharbitis nil seedlings. The photoperiodicinduction of one cotyledon was accomplished by wrapping it inaluminium foil for 13 to 15 h while the seedling remained inthe light. The presence of the other cotyledon in the lightblocked this inductive stimulus. The timing of its inhibitoryeffect suggested that its action was to block the expressionof the inductive stimulus, presumably at the shoot apex. Byvarying the area of the non-induced cotyledon parallel inhibitoryeffects were shown on export of stimulus and of ¹⁴C-labelledassimilate to the apex from the induced cotyledon. Thus, partof the inhibition was by interference with assimilate/stimulusco-transport in the phloem. However, an additional inhibitoryeffect was also evident and for this second component therewas no relationship between assimilate and stimulus transport.This latter inhibition was generated by brief light interruptionsof darkness given to one cotyledon only whilst the other waswrapped. The control treatment, removal of the unwrapped cotyledon,did not alter flowering compared to seedlings with intact, darkenedcotyledons. Thus, these studies show that the brief night interruptionsacted to trigger a photoperiodically sensitive inhibitor notto block induction. The implications of these findings are discussedin relation to models of time measurement in the photoperiodiccontrol of flowering. (Received March 20, 1989; Accepted November 16, 1989)     

Transport of shoot- and cotyledon-applied indole-3-acetic acid to Vicia faba root

To clarify the participation of indole-3-acetic acid (IAA) originatingfrom the shoot in root growth regulation and the mechanism ofIAA translocation from shoot to root, the movement of ¹⁴C-IAAwhich was applied to the epicotyl or the cotyledon of Viciafaba seedlings was investigated. The radioactivity of IAA appliedto the cotyledon moved faster to the root tip than that appliedto the epicotyl. On the basis of the effect of 2,3,5-triiodobenzoic acid on IAAmovement, a comparison with ¹⁴C-glucose movement and autoradiographicexamination, the nature of IAA movement was concluded to bepolar transport from the epicotyl to the basal part of the roots,while IAA movement from the epicotyl to the cotyledon, fromthe basal part of roots to the apical part, and from the cotyledonto the epicotyl and to the root took place in the phloem. Theradioactivity from ¹⁴C-IAA applied to the cotyledon accumulatedin lateral root primordia and vascular bundles. These factssuggest that IAA produced in cotyledons may participate in theregulation of Vicia root development. (Received December 21, 1979; )     

In Vitro Regeneration from Seedling Organs of Brassica oleracea var. italica Plenck cv. Green Comet I. Effect of Plant Growth Regulators

The calabrese cultivar Brassica oleracea var. italica cv. GreenComet was used in a study of the effects of exogenous hormoneson the growth and differentiation of seedling organs in vitro.Four types of explants were tested: hypocotyl segments, rootsegments, primary leaf discs and cotyledon discs. These explantswere incubated on media containing factorial combinations ofBAP x IBA, BAP x NAA, KN x IBA and KN x NAA (all at 0, 0.1,10 and 10.0mg l^–1). Hypocotyls were the most regenerativeexplants; shoot production was favoured by cytokinin: auxinratios greater than one and was decreased by IBA at 10 mg l^–1when callus was produced. Shoot formation from root explantsoccurred either in the absence of hormones or with low concentrations;no shoot was produced when any hormone was present at 10 mgl^–1. In contrast, shoot production from primary leaf diseswas favoured by high concentrations of both auxin and cytokininwith the combination of BAP and IBA the most effective. Shootproduction from cotyledon discs was sporadic with no consistentresponse on any auxin/cytokinin combination. After further experimentson the optimization of hormone concentration, the followingcombinations were chosen as allowing reliable regeneration:0.1 mg l^–1 BAP+0.1mg l^–1 IBA for hypocotyl segments,0.075 mg l^–1 KN +0.025 mg l^–1 IBA for root segments,and 5.0 mg l^–1 BAP+5.0 mg l^–1 IBA for leaf discs. Brassica oleracea var. italica, calabrese, tissue culture, seedling, auxin, cytokinin     

Kinetics of Phenylalanine Ammonia-Lyase and the Effect of L-{alpha}-aminooxy-{beta}-phenylpropionic Acid on Enzyme Activity and Radicle Growth in Germinating Lettuce Seeds

The effects of different concentrations of L--aminooxy-ß-phenyIpropionicacid (AOPP), an analog of L-phenylalanine, on the activity ofphenylalanine ammonia-lyase (PAL, EC 4.3.1.5 [EC] ) and the growthof radicles in 24 h old germinating lettuce (Lactuca salivaL.) seeds were investigated. AOPP causes a significant inhibitionof PAL activity in the seeds (85% inhibition at 10⁴ M). It alsocauses a stimulation of radicle growth at that concentration.The results show that the inhibition of PAL by AOPP may be dueto an irreversible binding of the inhibitor to the enzyme leadingto its inactivation. AOPP also inhibits ethylene biosynthesisin germinating lettuce seeds which could probably explain thestimulation of radicle growth in these seeds. The enzyme shows typical Michaelis-Menten kinetics. The K_m forL-phenylalanine is 4.2 x 10⁵ M. The enzyme does not show anytyrosine ammonia-lyase activity. Various substrate analogs suchas D-phenylalanine, p-fluorophenylalanine, ß-phenyllacticacid, tryptophan and the product of the enzyme reaction, trans-cinnamicacid, inhibit the enzyme competitively. A number of intermediatesand endproducts of the phenylpropanpid pathway, except chlorogenicacid, do not show any inhibition. ¹Scientific contribution number 1423 from the New HampshireAgricultural Experiment Station. (Received May 9, 1986; Accepted September 8, 1986)     

Intracellular pH Regulation in an Acidophilic Unicellular Alga, Cyanidium caldarium: 31P-NMR Determination of Intracellular pH

The intracellular pH of an acidophilic unicellular alga, Cyanidiumcaldarium, was determined as a function of external pH by ³¹Pnuclear magnetic resonance. The algal cells incubated underaerobic conditions or under anaerobic and illuminated conditionsmaintained the intracellular pH in the range from 6.8 to 7.0even when the external pH was changed from 1.2 to 8.4. Underanaerobic and dark conditions, however, the intracellular pHacidified at the acidic pH region of the external medium. Theacidified intracellular pH reversibly returned to neutral eitheron aeration or illumination. The results indicate that, in Cyanidiumcells growing in extremely acidic environments, an active H⁺efflux (H⁺ pump) which depends on metabolic activity (respirationor photosynthesis) is essential to maintain the intracellularpH at a constant physiological level against the passive H⁺leakage due to the steep pH gradient across the cell membrane. (Received March 19, 1986; Accepted July 17, 1986)     

The Inhibitory Effect of (2-Chloroethyl)-trimethylammonium Chloride on Chlorophyll and Protein Synthesis in Lettuce Cotyledons, and its Reversal by Potassium

Germinated seeds of Lactuca sativa (L.) were placed in Petri-dishesin (2-chloroethyl)-trimethyl-ammonium chloride (CCC; 0.005–0.05M), KN0₃ (0.01 M), and KC1 (0.01 M) solutions, and incubatedfor 2 or 5 days under continuous light. CCC strikingly arrestedchlorophyll accumulation, and retarded cotyledon growth relativelylittle. The retardant inhibited ¹⁴C-leucine incorporation intobulk proteins of the cotyledons. KN0₃ and KC1 promoted cotyledongrowth and chlorophyll synthesis per cotyledon by about 150per cent, and about doubled protein synthesis. Potassium saltscompletely reversed the inhibitory effects of CCC on chlorophylland protein synthesis. It is suggested that the inhibition ofgreening by CCC is dependent on a prior inhibition of proteinsynthesis.     

Potassium Transport in Non-Growing Corn Root Tissue as Affected by IAA and GA3

Experiments were done to determine if the spontaneous recoveryof non-growing segments of corn root (Zea mays L.) from excisioninjury is dependent on auxin. Washing the segments with 5 runindoleacetic acid (IAA) for 2 to 4 hours gave a small but significantincrease in K⁺ (⁸⁶Rb) influx, used here as a parameter reflectingrecovery of electrogenie H⁺-efflux pumping. This promotive effectwas obtained only after an hour of washing, and was sustainedby 100 nm gibberellic acid (GA₃). Any early responses to auxinwere obscured by an adverse reaction of the root cells to externalIAA which resulted in a transitory inhibition of H⁺ pumpingand K⁺ influx. Pretreatment of excised root tips with 10 µM IAA in thegrinding medium protected a plasmalemma-enriched fraction ofthe microsomes during isolation, giving increased uncoupler-sensitiveATPase activity. Non-growing root tissue thus shows three responses to auxin:an adverse reaction at the outer surface of the plasmalemmawhich blocks H⁺ pumping; a protective or restorative effecton the H⁺-ATPase; an increased capacity for K⁺ influx duringthe developmental phase of washing, which is augmented by thepresence of GA₃. (Received March 31, 1986; Accepted September 8, 1986)     

An Analysis of Seed Development in Pisum sativum: IV. COTYLEDON CELL POPULATIONS IN VIVO AND IN VITRO

A method for quantifying changes in the cell population of Pisumsativum cotyledons during development is described. The methodis based on determining the frequency distribution for cellarea following the random sampling of a single-cell suspensionof cotyledon cells. The population profile of these cells changedprogressively and systematically from a single population, similarin size to meristematic cells, found in embryos less than 3.0mg in fresh weight, to a bimodal population in embryos greaterthan 100 mg fresh weight. This method was used to compare embryosof similar size from two genotypes near-isogenic except forgenes at the r locus. No significant differences were foundbetween the cell population profiles of embryos up to 30 mgfresh weight. However, a significant difference was found betweenembryos with fresh weights of 100 mg, the wrinkled (rr) linehaving a higher mean and maximum cell area (2 951 µm²and 9 240 µm² respectively) than the round (RR) line (2591µm²and 6470 µm²respectively). Comparisons were alsomade between cotyledon cell populations from round (RR) embryosgrown in vivo and in vitro. The most obvious differences werethe higher mean and maximum cell size of the large cell populationof in vitro grown embryos which were twice those found in vivo.Embryos grown to either 30 mg or 100 mg fresh weight in vitrohad a much greater proportion of large cells in the populationwith a corresponding reduction in total cotyledon cell number,compared with similar sized embryos grown in vivo. These data suggest that comparisons between different genotypes,or, between cultured and in vivoembryos, based on morphologicalsimilarities between embryos, may be invalid and subject tomisinterpretation. Key words: Pea, seed development, cell population     

Changes in Steady-State Photosynthetic 14C-Incorporation into cellular Metabolites of Chlorellax pyrenoidosa during Transitions from High to Low Irradiance

Changes in the levels of ¹⁴C-labelled metabolites were monitoredin Chlorella pyrenoidosa cells during a transition from highto low irradiance, i.e., from 700 to 430 µmol quanta (400–700nm) m^–2 s^–1. Chlorella cells assimilated ¹⁴CO₂ photosynthetically(steady-state ¹⁴C-labelling) for 12 min at the high irradianceand then 10 min at the low irradiance. With the transition tolow light, the level of ¹⁴C-labelled ribulose 1,5- bisphosphate(RuBP) did not decrease, even though the rate of total ¹⁴C-incorporationdecreased by 80%. The data suggest that RuBP carboxylase deactivatesrapidly (within 1 or 2 min) on exposure to low light, causingRuBP pool sizes to be maintained (or even increased) in spiteof a decreased rate of RuBP regeneration. There was also evidenceof light modulation of other enzymes, including some enzymesinvolved in sucrose synthesis. The rate of sucrose synthesisdecreased with decrease in light intensity while the level ofuridine diphosphoglucose increased, but within a few minutes,both returned to their former levels. ¹Present address: Chemical Biodynamics, Lawrence Berkeley Laboratory,Building 3, 1 Cyclotron Road, Berkeley, CA 94720, U.S.A. (Received March 8, 1986; Accepted June 25, 1986)     

Chlorophyll-Protein Complexes Associated with Photosystem I Isolated from the Green Alga, Bryopsis maxima

Six chlorophyll (Chl)-protein complexes associated with photosystemI (CPla), and the PS I reaction center complex (CPl) were isolatedfrom the thylakoid membranes of the green alga, Bryopsis maxima,by SDS-polyacrylamide gel electrophoresis. CPla had four polypeptides(22, 24, 25, 26 kDa) in addition to the 67 kDa polypeptide ofCPl. These complexes may thus possibly be a combination of CPland antenna complexes for PS I. Six CPla showed almost the sameoptical properties, with absorption maxima at 650 and 677 nmand contained carotene and a small amount of xanthophylls. TheChl a/b ratios of these CPla were about 2, while that of CPlwas 14. CPla showed a fluorescence emission maximum at 695 nm;its excitation spectrum had peaks at 438, 470 and 540 nm, correspondingto the absorption maxima of Chl a, Chl b, xanthophylls, respectively.An antenna complex free of CPl has been detected in some plantsbut was not found in the present alga. ¹Present address: Department of Botany, The University of Adelaide,Adelaide, S.A. 5001, Australia (Received April 17, 1986; Accepted June 26, 1986)     

Nucleoside triphosphate diphosphatase from germinated pea cotyledon chromatin

Nucleoside triphosphate diphosphatase purified from germinatedpea cotyledon chromatin catalyzed the phosphohydrolysis of nucleosidetriphosphate or diphosphate to nucleoside monophosphate andPi. This enzyme differed from the cytoplasmic phosphatase inthe cotyledon; it had no activity against the other phosphateesters. The enzyme was activated by Mg²⁺ and Mn²⁺, and had apH optimum of 7.0 and a Km of 9.6 ? l0^–8 M for GTP, 1.2? l0^–5M for UTP, 2.7 ? l0^–5 M for CTP and 1.7 ?l0^–4M for ATP. Pyrophosphate inhibited the activity withboth nucleoside triphosphate and diphosphate as substrates.The degree of the inhibition by pyrophosphate to each nucleosidetriphosphate as substrate was the reverse of the affinity ofthe substrate. ¹ Present address: Institute for Agricultural and BiologicalScience, Okayama University, Chuo, Kurashiki, Okayama 710, Japan. (Received April 26, 1979; )     

Analysis of Rapid Light-Induced Potential Change in Cells of Chara corallina

Perfused Chara cells were used to measure the rapid light-inducedpotential change (rapid LPC) caused by activation of a K⁺ channelin the plasma membrane through photosynthesis in the presenceof various photosynthetic inhibitors. The rapid LPC was inhibitedby DCMU but recovered on addition of phenazinemethosulfate (PMS)in the presence of DCMU. Carbonylcyanide m-chlorophenylhydrazone(CCCP) stimulated the rapid LPC. DCCD partially inhibited therapid LPC with a partial inhibition of oxygen evolution. Itis concluded that both cyclic and noncyclic electron flows arecoupled with the rapid LPC. To understand the mechanism of K⁺ channel activation by photosyntheticelectron flow, the rapid LPC was measured under continuous internalperfusion. It was suggested that a diffusible substance wasnot released from chloroplasts, since vigorous continuous perfusiondid not inhibit the rapid LPC. The suggestion that the rapid LPC is caused by changes in surfacecharge density of chloroplasts was supported by the fact thatthe rapid LPC was inhibited by increasing the ionic strengthof the perfusion medium. (Received February 28, 1986; Accepted April 30, 1986)     

Action Spectrum in Ultraviolet and Blue Light Region for the Inhibition of Red-Light-Induced Spore Germination in Adiantum capillus-veneris L.

The action spectrum for the inhibition of red-light-inducedgermination of spores in the fern Adiantum capillus-veneriswas determined between 250 and 500 nm using the Okazaki largespectrograph. When monochromatic lights were given after red-lightirradiation, two prominent peaks for inhibition of spore germinationwere observed at 275 and 440 nm and a minor peak at ca. 390nm. ² Permanent address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan.     

Photoreversible and photoirreversible absorbance changes in the red and far-red spectral regions in Zea primary roots

When 2-mm apical segments of the primary roots of Zea mays L.(cv. Golden Cross Bantam 70) were irradiated successively withred and far-red light, a photoirreversible absorbance decreasewas separated from the red far-red reversible absorbance changetypical of phytochrome. The difference spectrum of the reversiblechange showed maximum absorbance changes at 666 and 730 nm,while the photoirreversible change induced by red light showeda maximum decrease at 640 nm. The photoreversible absorbancechange was linearly proportional to the fluence of red lightbetween 1 and 6 J m^–2, while the photoirreversible absorbancechange was proportional to its logarithm. Red light of approximately6 J m^–2 induced 50% of the maximum photoirreversible absorbancechange at 640 nm but only about 25% of the maximum photoreversibleabsorbance change. Moreover, no effect of ascorbate on the twoabsorbance changes was observed. ¹Faculty of Education, University of Yamagata, Yamagata 990,Japan. (Received November 2, 1980; )     

Photoreversible and photoirreversible absorbance changes in the red and far-red spectral regions in Zea primary roots

When 2-mm apical segments of the primary roots of Zea mays L.(cv. Golden Cross Bantam 70) were irradiated successively withred and far-red light, a photoirreversible absorbance decreasewas separated from the red far-red reversible absorbance changetypical of phytochrome. The difference spectrum of the reversiblechange showed maximum absorbance changes at 666 and 730 nm,while the photoirreversible change induced by red light showeda maximum decrease at 640 nm. The photoreversible absorbancechange was linearly proportional to the fluence of red lightbetween 1 and 6 J m^–2, while the photoirreversible absorbancechange was proportional to its logarithm. Red light of approximately6 J m^–2 induced 50% of the maximum photoirreversible absorbancechange at 640 nm but only about 25% of the maximum photoreversibleabsorbance change. Moreover, no effect of ascorbate on the twoabsorbance changes was observed. ¹Faculty of Education, University of Yamagata, Yamagata 990,Japan. (Received November 2, 1980; )     

Thermal Properties of Membrane Lipids from two Cyanobacteria, Anacystis nidulans and Synechococcus sp.

The composition and positional distribution of fatty acids inmonogalactosyldiacylglycerol, digalactosyldiacylglycerol, phosphatidylglyceroland sulphoquinovosyldiacylglycerol from two cyanobacteria, Anacystisnidulans and Synechococcus sp. grown at 25°C have been determinedand compared with measurements of the phase separation temperaturesof the lipids. Only monogalactosyldiacylglycerol in Anacystisand sulphoquinovosyldiacylglycerol in Synechococcus showed phaseseparation temperatures above 0°C. The phase transitiontemperature of a sample of sulphoquinovosyldiacylglycerol containingover 90% of the dihexadecanoyl molecular species has been determinedto be 43°C for the Na⁺ salt and 38°C for the Mg⁺⁺ salt. ^*Deceased. September 14, 1986. (Received June 25, 1986; Accepted August 25, 1986)