Cytokinin control in subcellular localization of indoleacetic acid oxidase and peroxidase

IAA oxidase and peroxidase were found in all subcellular fractions of tobacco callus cells. The bound and cytoplasmic fractions differed greatly in IAA oxidase/peroxidase ratio and in isoperoxidase composition. The IAA oxidase/peroxidase ratio was particularly high in the plasma membrane-rich fraction. Kinetin had profound effects on IAA oxidase and peroxidase. The appearance of fast migrating isoperoxidases in response to 0·2 μM kinetin was found only in cytoplasmic, plasma membrane and ribosome-rich fractions; a high concentration of kinetin inhibited their formation. High kinetin concentrations also lowered the specific activity of IAA oxidase and peroxidase in all subcellular fractions, but the effect was much greater on peroxidase than on IAA oxidase, thus resulting in a drastic increase in IAA oxidase/peroxidase ratio. Evidently the activities of IAA oxidase and peroxidase were not equivalent and should be considered separately.     

Isoperoxidase Patterns in Tobacco Pith and their Alteration Following Tissue Excision

Previous work has shown that young tobacco pith cells containlow peroxidase activity resolvable into two anodic iso-zymesby electrophoresis at pH 8.3 on starch gel. During normal cellenlargement and ageing in situ, these two isoenzymes increaseslowly in activity. By contrast, within 24 h after excisionof pith, high activities appear in several apparently new anodicand cathodic isoperoxidases. This development of new isoperoxidasesis prevented by physiological levels of IAA and by inhibitorsof RNA and protein synthesis. The present work describes additional quantitative isoperoxidasegradients, both vertical and horizontal, in the pith. Severaladditional isoenzymes, some of which are excreted into the mediumduring incubation, have been detected by improved electrophoretictechniques. Enzyme leakage can be prevented by incubation inmoist chambers and by auxin application through terminally placedagar blocks. With such techniques, new peroxidases form preferentiallynear cut surfaces within 12 h under aerobic conditions. Tissuesperipheral to the pith retard the development of new peroxidaseactivity. IAA in agar blocks applied terminally to a cut pithcylinder retards peroxidase formation mainly at the base. Theregulation of the quantity and quality of isoperoxidases seemsto be correlated with certain morphogenetic phenomena.     

The effect of kinetin on the indoleacetic acid level and indoleacetic acid oxidase activity in roots of young plants

Kinetin treatment increased the extractable IAA content in roots of young plants of Phaseolus vulgaris L., Zea mays L. and Avena sativa L. The highest increase was obtained with roots of beans and the lowest with oat roots. Maize was intermediate between these two species. Kinetin treatment decreased the activity of IAA-oxidase but the correlation between the decrease of the activity of this enzyme and the increase in the level of IAA was not good. The decrease of the oxidase activity was greatest in oat roots where kinetin had very little effect on the IAA level, and was rather small in bean roots, where kinetin treatment significantly increased the IAA level.     

Hormonal control of isoperoxidases in lentil embryonic axis

Detachment of the cotyledons from the lentil (Lens culinaris Med.) embryonic axis causes in the latter an increase in total peroxidase activity which is shown to be due to enhancement of specific cathodic isoperoxidases. Kinetin treatment of attached or detached axes promotes activity of essentially the same cathodic isoperoxidases. In addition kinetin enhances the activity of two anodic peroxidases and represses specifically that of a cathodic one. Abscisic acid inhibits the production of all isoenzymes in the presence or absence of kinetin. Cytokinin and abscisic acid actions are discussed in relation to the nature of a wounding hormone and the role of natural inhibitors in cotyledons during germination. Indoleacetic acid stimulates the activity of certain isoenzymes which are also stimulated by kinetin, whereas in others the effects of the two hormones are different. Specific inverse effects of indoleacetic acid and kinetin are demonstrated on the two most cathodic isoperoxidases. Indoleacetic acid-kinetin interactions on the cathodic isoperoxidases have been found in the literature and are discussed as a possible mechanism for explaining interactions of the two regulators on growth and other physiological processes.     

HORMONAL CONTROL OF PEROXIDASE ACTIVITY IN CULTURED PELARGONIUM PITH

Freshly excised Pelargonium pith tissue lacks peroxidase activity toward guaiacol or benzidine, but it develops such activity within 24–36 hr in aseptic culture. All the activity is manifested as a single enzyme moving toward the cathode during electrophoresis on starch gel at pH 9.0. This development of peroxidase activity is at first (up to ca. 50 hr in culture) inhibited and later (ca. 100–150 hr in culture) promoted by IAA. This dual effect of IAA resembles that previously reported for specific isoperoxidases in tobacco pith cells. Kinetin alone also inhibits peroxidase formation, but in the presence of IAA those concentrations which enhance growth enhance peroxidase formation as well.     

Control of Hypocotyl Hook Angle in Phaseolus mungo L. Role of Growth Substances

Effects of growth hormones on the hook angle and light responseof Phaseolus mungo L. hypocotyl hooks are described and theresults are discussed with reference to the functions of otherparts of the seedling in controlling the growth and shape ofthe hook. Apically applied IAA (indolyl acetic acid) prevented hook openingin decapitated seedlings in the dark and in all the red-irradiatedseedlings. [¹⁴C]IAA experiments showed that only a small quantityof IAA (2–6 ?g per hook) was required to produce theseeffects, and that transport of IAA through the hook was negligibleand unaffected by red irradiation. ABA (abscisic acid) had little effect on the hooks or theirlight response. Applied ethylene and IAA-induced ethylene slightly closed thehooks, but only slightly reduced light-induced opening. IAAreduced the effect of ethylene in the dark, but after irradiationthe hooks appeared more sensitive to the ethylene in the presenceof IAA, resulting in light-induced hook closure. Basally applied kinetin (6-furfurylaminopurine) prevented decapitatedhooks from opening in the dark, especially when GA₃ (gibberellicacid) was also present. Some combinations of kinetin and GA₃(with high kinetin concentrations) also prevented light-inducedopening, but combinations with lower kinetin concentrationsallowed almost as much opening as was found in intact hooks. It is proposed that the terminal parts act by regulating thesupply of cytokinins and gibberellins from the basal parts,and that IAA does not mediate this funotion in this species. The results are compared with those reported for other species.     

Indole-3-Acetic Acid Control on Acidic Oat Cell Wall Peroxidases

Incubation of oat coleoptile segments with 40 μm indoleacetic acid (IAA) induced a decrease of 35–60% in peroxidase activity at the cell wall compartment. Treatment with IAA also produced a similar decrease in the oxidation of NADH and IAA at the cell wall. Isoelectric focusing of ionic, covalent, and intercellular wall peroxidase fractions showed that acidic isoforms (pI 4.0–5.5) were reduced preferentially by IAA treatment. Marked differences were found between acidic and basic wall isoperoxidases in relation to their efficacy in the oxidation of IAA. A peroxidase fraction containing acidic isoforms oxidized IAA with a V _max/s_0.5 value of 2.4 × 10⁻² min⁻¹· g fw⁻¹, 4.0 times higher than that obtained for basic peroxidase isoforms (0.6 × 10⁻² min⁻¹· g fw⁻¹). In contrast, basic isoforms were more efficient than acidic isoperoxidases in the oxidation of coniferyl alcohol or ferulic acid with H₂O₂ (5.6 and 2.1 times, respectively). The levels of diferulate and lignin in the walls of oat coleoptile segments were not altered by treatment with IAA. The decrease in cell wall peroxidase activity by IAA was related more to reduced oxidative degradation of the hormone than to covalent cell wall cross-linking. Received November 1, 1998; accepted December 14, 1998     

Changes in chloroplast peroxidase activities in relation to chlorophyll loss in barley leaf segments

Total peroxidase activity increased during senescence of excised barley ( Hordeum vulgare L. cv. Kashimamugi) leaves. Kinetin treatment furter increased total peroxidase activity but repressed chlorophyll degradation in excised barley leaves. When isoperoxidases were extracted from barley leaf segments. 4 cationic and 4 anionic isozymes were found in polyacrylamide gel electrophorests during leaf senescence. The chloroplasts contained only two cationic isoperoxidase activities. One (designated C4) was repressed by kinetin. and the other (C3) was increased by kinetin. Glucosamine, which also repressed the degradation of chlorophyll, completely repressed C4 activity but did not affect C3 activity. The induction with senescence, and the repression with kinetin and glucosamine, suggest chat chloroplast isoperoxidase C4 may function as a chlorophyll-degrading enzyme during barley leaf senescence.     

Effect of Light and Benzyladenine on Dark-Treated Growing Rice (Oryza sativa) Leaves II. Changes in Peroxidase Activity

Changes in peroxidase activity were studied in the attachedfirst leaf of dark-treated Oryza sativa L. cv. Bala seedlingsin response to benzyladenine and light treatments during laterperiods of leaf growth, prior to maturation. Darkness causeda mild decrease in peroxidase activity; but in illuminated leaves,the enzyme activity was stable at all times. There was a sharprise in peroxidase activity in dark-treated leaves upon lightor benzyladenine application, irrespective of the time of treatment.Benzyladenine treatment to illuminated leaves also caused arise in peroxidase activity. Exogenous hydrogen peroxide, glycolateand amizol resulted in a rise in peroxidase activity, whichwas further enhanced by benzyladenine treatment in both lightand dark incubated leaves. Proline maintained chlorophyll levels,whereas hydroxyproline caused chlorophyll degradation. Benzyladenineenhanced the proline effect and counteracted the hydroxyprolineeffect on chlorophyll. Both proline and hydroxyproline increasedperoxidase activity in the leaves of light and dark incubatedseedlings, and the enzyme activity further increased after benzyladeninetreatment. (Received December 7, 1984; Accepted May 8, 1985)     

The Effects of Growth Substances on the Growth of Tapinanthus bangwensis (Loranthaceae) in vitro

The growth response of Tapinanthus bangwensis (Engler and K.Krause) Danser to exogenous growth substances, indole-3yl-aceticacid (IAA), kinetin, and gibberellic acid (GA₃), not only varieswith the concentrations of each of these substances in the mediumbut follows a similar trend in each case. Kinetin, IAA, andGA₃ were found to enhance growth but not by very appreciableamounts, kinetin being the most effective growth promoter. Highconcentrations of these substances (5.0 and 10.0 ppm) exceptGA₃ were toxic to the parasite. Gibberellic acid induced theproduction of accessory leaves at all the concentrations used.Kinetin was also effective in inducing accessory leaves butonly at a concentration of 1.0 ppm. Some of the lower concentrationsof the growth substances affected significantly some aspectsof the parasite's growth and development. Nevertheless, thegrowth in vitro was still slower than that in vivo.     

Auxin-gibberellin relationships in their effects on hypocotyl elongation of light-grown cucumber seedlings VI. Promotive and inhibitory effects of kinetin

The interaction of kinetin with IAA and GA₃ on the elongationof hypocotyl sections of Cucumis sativus L. cv. National Picklingwas studied. Kinetin in the concentration range of 10^–7M to 10^–4 M markedly inhibited IAA-induced elongation,while in a lower range from 10^–10 M to 10^–8 M, itsynergistically enhanced IAA-induced elongation. Kinetin alonein this range had no effect. A 5-to 15-min pulse treatment seemsenough to induce the maximum effect for both inhibition andpromotion. Since the magnitude of the maximum inhibition dependedon the concentration and not on the duration of treatment, thereaction in the cell caused by kinetin seemed to be completedwithin a short period. Washing of the sections with distilledwater after kinetin treatment (30 min) did not significantlyeliminate the kinetin effect. This probably indicates that thebinding of kinetin molecules to a supposed acceptor is not reversible.Interaction of kinetin with GA₃ in their pretreatment effectson IAA-induced elongation shows that in the inhibitory concentrationrange, the kinetin effect was partly overcome by GA₃, and thatin the promotive range, the magnitude of the enhancement wasdetermined by kinetin regardless of the presence of GA₃. Theeffect of kinetin seems to dominate over that of GA₃ indicatingthat the modes of their pretreatment effects differ from oneanother. (Received June 24, 1977; )     

Synergistic effect of kinetin on IAA-induced ethylene production

Kinetin has a significant synergistic effect on IAA-inducedehtylene production in hypocotyl sections of eitolated mungbean(Phaseolus mungo L.) seedlings when it is administered immediatelyafter cutting. If the addition of kinetin was delayed, it becameless effective. Kinetin-pretreated sections followed by incubationeither in IAA or in IAA plus kinetin produced ehtylene essentiallyat an identical rate to the one treated with IAA plus kinetinat zero time. However, the buffer-preincubated sections followedby incubation in IAA or in IAA plus kinetin shows a much reducedrate of ehtylene production as compared with the one treatedwith IAA or with IAA plus kinetin at zero time, respectively.These results suggest that kinetin is required for the maximalstimulation of ethylene production only during the early incubationperiod while IAA is required continuously throughout the incubation;the removal of IAA from the IAA-treated tissues causes an immediatecessation of ethylene production. ¹Present address: Mann Laboratory, Department of Vegetable Crops,University of California, Davis, California 95616, U.S.A. (Received May 10, 1973; )     

The effect of 4-chlororesorcinol on the endogenous levels of IAA,ABA and oxidative enzymes in cuttings

Treatment of bean cuttings with 4-chlororesorcinol (4-CR), known to increase the number of roots and extend their distribution, prevented the accumulation of free indol-3-yl-acetic acid (IAA) in the hypocotyls within 24 h after cutting preparation. In mung bean there was no change in the distribution (upper half vs. 1 ower half of the hypocotyl) of IAA within the hypocotyl as a result of the treatment. In bean cuttings the treatment with 4-CR prevented the accumulation of IAA in the bottom of the cutting. Oxidation of IAA as a measure of IAA oxidase activity in bean was enhanced appreciably by 4-chlororesorcinol. The level of abscisic acid in mung bean, on the other hand, remained 3–4 fold higher than in the control, yet still about 50% lower than the zero time level. In untreated mung bean cuttings the activity of peroxidase increased after cutting preparation. In contrast, the activity of peroxidase in 4-Cr-treated cuttings was consistently lower. In order to relate to the effect of exogenously applied auxin the level of peroxidase was measured also in indol-3-yl-butyric acid-treated cuttings. The overall peroxidase activity in IBA-treated cuttings was not affected. However, when assaying for the different isozymes the drop in peroxidase activity was most evident in the inducible basic isoperoxidases both in 4-CR and IBA treatments. It appears that the exposure to 4-CR exerts an effect that is similar to that of exogenously applied auxin, affecting the activity of basic peroxidases and enhancing the oxidation of endogenous IAA, thus allowing the organization of the primordia.Abbreviations ABA - abscisic acid - 4-CR - 4-chlororesorcinol - IAA - indol-3-yl-acetic acid - IBA - indol-3-yl-butyric acid     

Effects of N-1-naphthylphthalamic acid on the growth and bud formation of tobacco callus grown in vitro

The effect of N-1 -naphthylphthalamic acid (NPA), indole-3-aceticacid (IAA) and kinetin on callus growth and bud formation wasstudied mainly by a tobacco callus culture method. Callus producedfrom Nicotiana tabacum var. Wisconsin 38 was used as the testplant material. Callus growth on nutrient agar containing 2mg/liter of IAA was promoted by NPA added at a concentrationof 0.5 mg/liter with 0.4 mg/liter of kinetin or by NPA addedat 5 mg/liter in the absence of kinetin. At a high concentrationof 50 mg/liter, however, NPA inhibited growth on the mediumcontaining 2 mg/liter IAA and no kinetin. Kinetin reduced thisNPA inhibition. In the presence of 0.4 mg/liter kinetin and2 mg/liter IAA, when the concentration of NPA was 50 mg/liter,buds were initiated after calluses were grown on the test mediumfor 7 weeks in dim light, but no buds formed when NPA was omittedfrom the above medium. The control of callus growth and bud initiation is based onthe active ratio of auxin (IAA) to cytokinin (kinetin) in themedium and NPA added to the medium can promote or inhibit callusgrowth and induce bud formation. Therefore, it is proposed thatNPA can itself reduce auxin activity or enhance cytokinin activityand hence change the active ratio of the two regulators. NPAmay enhance the activity of cytokinin (here supplied as kinetin)but cannot substitute for it. ¹Present address: Department of Biology, Wisconsin State University,Oshkosh, Wisconsin 54901, U. S. A. (Received March 10, 1969; )     

Studies on senescing tobacco leaf disks with special reference to peroxidase

Summary Senescing tobacco leaf disks were treated with hydroxyproline (hypro). Chlorophyll breakdown and -amino-nitrogen increase were partially inhibited, but the decrease in soluble protein was stimulated. The normal increase in absolute acid-phosphatase activity was reversed, but the increase in peroxidase activity occurred although after a lag phase. Kinetin inhibition of senescence was reversed by hypro and kinetin actually reinforced the effects of hypro. Proline only partially overcame the effects of hypro plus kinetin. In the presence of kinetin, proline considerably stimulated the increase in peroxidase activity.Discussed are the way in which hypro may be acting, and the possibility of peroxidase being involved in proline hydroxylation.     

Kinetin-induced Formation of Gametophores in Dark Cultures of Ceratodon purpureus

The effects of various concentrations of kinetin on the developmentof gametophytes were studied in light and dark cultures of Ceratodonpurpureus in liquid media. There was no bud formation in eitherlight or dark control cultures. Buds were produced under theinfluence of kinetin in light as well as in dark cultures, theeffect of a given concentration of kinetin being a little strongerin the light. Kinetin has been thus found to be an essential factor of budformation in a moss which hardly produces gametophores evenin the light. This is believed to be the first report of gametophore formationin the dark since Keil's short communication in 1949. It alsorepresents a new example replacing a morphogenetic light stimulusby kinetin.     

Changes in Paramagnetic Manganese (Mn2+) and Related Enzyme Activities in Isolated Cucumber Cotyledons During Growth: II. EFFECT OF 6-FURFURYLAMINOPURINE

Changes in the activities of IAA oxidase, peroxidase, ascorbicacid utilization (AAU), and in the level of paramagnetic manganese(Mn²⁺) have been studied during kinetin-induced growth of theisolated cucumber cotyledons in light or in dark. In kinetin-treatedcotyledons exposed to light, inhibition in the level of paramagneticmanganese corresponds with an enhancement in IAA oxidase activity.The level of paramagnetic manganese shows an inverse correlationwith IAA oxidase activity. In darkness the level of Mn²⁺ doesnot show the same correlation with IAA oxidase activity as inthe light. Kinetin stimulates peroxidase activity both in thelight and in darkness. Enhancement of IAA oxidase activity andno corresponding change in the level of paramagnetic manganeseindicates that the oxidation of IAA in dark-grown, kinetin-treatedcotyledons is brought about by peroxidase. It appears that thephenolic cofactors required for the oxidation of manganese andIAA may be limiting in kinetin-treated cotyledons in darkness.Thus in the light, IAA oxidation seems to be brought about byperoxidase as well as manganese, whereas in darkness it is mediatedby peroxidase alone. Increase in IAA oxidase activity duringkinetin-induced growth of the isolated cotyledons is incompatiblewith the idea that increased IAA oxidase activity would limitthe availability of auxin for growth. Kinetin does not mimicthe action of light on IAA oxidase activity; on the contrary,it removes the inhibitory effect of light on IAA oxidase activityprobably through the synthesis of IAA oxidase activators.     

Senescence in Detached Oat Leaves I. Changes in Free Amino Acid Levels

Changes in the levels of free amino acids have been measuredduring light and dark senescence of oat leaf segments. Leaveswere aged either on water, 5 ppm kinetin or 30 ppm abscisicacid. The patterns with which levels of individual amino acidschange differ a great deal in leaves senescing either in darkor light, signifying that different mechanisms may regulateoat leaf senescence in light and dark. Levels of serine andmost of the other amino acids that increase substantially duringdark senescence of oat leaves change parallel to mitochondrialrespiration. Kinetin depresses the rise in amino acids justas it does with respiration in the dark. The synthesis of serineproteases does not seem to be limited by the availability ofendogenous serine. The levels of glutamine increase dramaticallyin leaves kept in light (ca. 2,200% of initial value within7 days) but only a little in the dark, which may reflect a possiblerole of photorespiration during the senescence of oat leavesin the light. Abscisic acid enhances the release of amino acidsmore strongly in the light than dark. The senescence promotingeffect of abscisic acid in the light seems to bring about changesin amino acid levels similar to those that appear in leavessenescing on water in the dark. ¹ Present address: C.F. Kettering Research Laboratory, 150 EastSouth College Street, Yellow Springs, Ohio 45387, U.S.A. (Received June 24, 1981; Accepted October 30, 1981)     

Effects of Plant Growth Regulators on Grain-filling and Yield of Rice

Effects of three phytohormones (IAA₁, GA₃ and kinetin) on grain-fillingand the pattern of ³²P translocation from individual leavesto grains were studied at intervals of 7 days during the progressof reproductive development of rice (Oryza saliva L. cv. Jaya).The plants were sprayed with 100 µg ml^–1 aqueoussolutions of the hormones at 100 days, when the plants wereentering the reproductive stage. Kinetin produced a pronouncedeffect on grain-filling as well as on ³²P mobilization fromindividual leaf to grains and increased yield, possibly by increasingleaf longevity. GA₃ and IAA also increased the grain-fillingand ³²P mobilization significantly over control but the effectswere less marked than those of kinetin. Oryza sativa L., rice, grain yield, translocation, growth regulators, gibberellic acid, indol-3-yl acetic acid, kinetin     

Variations de quelques aclivités enzymatiques (peroxydase, catalase, AIA-oxydase) et de la teneur en polyphénols au cours de la germination de I'Orge. Influence de la kinétine

Peroxidase catalase, IAA-oxidase and polyphenol content of growing barley coleoptile. Effect of kinetin. - Kinetin strongly inhibits root and coleoptile growth of germinating barley in the dark. Treated coleoptiles become senescent before the untreated ones. Soluble proteins content, peroxidase, catalase and IAA-oxidase activity were greatly increased in treated coleoptiles while the level of polyphenols was reduced. These biochemical effects joined with the other property of kinetin to diminish α-amylase synthesis in the endosperm are discussed in relation to growth and in connection with the classic view of a cytokinin retarded senescence.