Polarity of Production of Polyphenols and Development of Various Enzyme Activities in Cut-injured Sweet Potato Root Tissue

Investigation of polyphenol production in cut-injured sweet potato (Ipomoea batatas Lam. cv. Kokei 14) roots by histochemical and quantitative methods showed that polyphenols were produced in striking amounts in the proximal side of the tissue pieces (2 cm thick), but only in small amounts in cells of the distal side. In response to cut injury, formation of the enzymes related to polyphenol biosynthesis, phenylalanine ammonia-lyase and trans-cinnamic acid 4-hydroxylase, was also pronounced in the proximal side of the tissue pieces and slight in the distal side. The similar polarity was observed in the development of activities of various enzymes, such as NADPH-cytochrome c oxidoreductase, acid invertase, peroxidase, o-diphenol oxidase, and cytochrome c-O₂ oxidoreductase. Acropetal development of polyphenol contents and of various enzyme activities may be related to the acropetal movement of indoleacetic acid (IAA) in roots of various plants. Treatment of the distal surface of tissue pieces with IAA or 2,4-dichlorophenoxyacetic acid caused polyphenol production but treatment with gibberellic acid, abscisic acid, kinetin, or ethylene had little effect. The results suggest that IAA may play a role in the metabolic response to cut injury.     

Studies on the change of the respiratory system in roots in relation to the growth of plants II. Activity of iron-containing oxidases in roots of cereal crops with the aging of plants

Distribution of iron-containing oxidases in aging nodal rootsof rice and wheat was studied. Activities of cytochrome c oxidase(1.9.3.1 [EC] , cytochrome c : O₂ oxidoreductase), catalase (1.11.1.6 [EC] ,H₂O₂: H₂O₂ oxidoreductase) and peroxidase (1.11.1.7 [EC] , donor:H₂O₂ oxidoreductase) in wheat roots were comparatively higherthan were those in rice roots at corresponding stages. Cytochromec oxidase in roots remained active throughout the lives of therice and wheat crops. In rice roots, catalase seemed to playa distinct role around the panicle formation stage. Decay ofcatalase activity took place earlier than did that of peroxidaseand cytochrome c oxidase activities. In wheat roots similarenzyme activity changes were not observed. Data may suggestthat the high activity of iron containing oxidases at the panicleformation stage (I) may be chiefly due to catalase activityin rice roots. ¹Paper presented at the 14th Annual Meeting of the Society ofthe Science of Soil and Manure, Japan (1968). (Received November 21, 1968; )     

Polar transport and content of indole-3-acetic acid in wounded sweet potato root tissues

In roots of sweet potato (Ipomoea batatas Lam. cv. Kokei 14),the metabolic response to wounding was remarkable in the proximalside and developed in the acropetal direction. We assumed thatthe polarity resulted from the increase in polar movement ofindoleacetic acid (IAA) (1977, Plant Physiol. 60: 563–566).Transport of IAA and change of the IAA level in the woundedtissue of sweet potato roots were investigated. Transport ofthe label from ¹⁴C-IAA was obviously polarized in the acropetaldirection. ¹⁴C-IAA administered to the wounded tissue was mainlymetabolized into two conjugates of IAA. The amount of IAA inthe wounded tissue, determined by the spectrofluorometric method,increased about 3-fold after 18 hr of incubation prior to thedevelopment of activities of some enzymes. The increase in IAAcontent was not affected with aseptic incubation, therefore,the possibility of IAA production by microorganisms on the woundedtissue was excluded. The results obtained strongly support ourhypothesis that IAA plays an important role in the metabolicresponse to wounding. (Received May 2, 1979; )     

Indole-3-acetic acid Metabolism in Pea Seedings: A Comparative Study using Carboxyl-and Ring-Labelled Isomers

Etiolated pea plants, grown aseptically, were treated with eitherring- or carboxyl-labelled indole-₃-acetic acid (IAA), and subsequentlythe carbon dioxide which had been released, the residual culturesolution, and the dissected plant parts were examined for radioactivecompounds. Although some breakdown of IAA was detected in theabsence of plants, much more decarboxylation occurred when theywere present. The only metabolite found in any quantity in vivowas IAA-aspartate. Very small amounts of indole-₃-aldehyde,and of other substances identified only tentatively, were alsoobserved. A further substance, possibly N-hydroxy indole-₃-aceticacid, was found in the seedlings as well as in the culture medium;its presence in vivo is unlikely to have any metabolic significance.The paucity of products in the seedlings suggests that rapidbreakdown of IAA does not occur in vivo.     

Comparative Effects of lndole-3-acetic acid, Abscisic acid, Gibberellic acid and 6-Benzylaminopurine on the Dark- and Light-induced Pulvinar Movements in Cassia fasciculata Michx

Effects of plant hormones were examined on the dark- and light-inducedmovements of Cassia fasciculata. Indole-3-acetic acid (IAA),gibberellic acid (GA₃) and 6-benzylaminopurine (6-BAP) inhibitedthe scotonastic movement whereas abscisic acid (ABA) enhancedit. After brief treatments (5 to 30 min), the ABA effect wasinhibitory rather than promotional. Hormonal treatment in theacidic range gave the best physiological response for ABA, butthe greatest efficiency of IAA, GA₃ and 6-BAP was obtained withpH values close to neutrality. Three to 5 h were needed beforeexpression of the physiological effect triggered by GA₃ and6-BAP, while 5 min treatments were sufficient for IAA and ABA.Light-induced movements were largely enhanced by IAA and slightlyby GA₃ but inhibited by 6-BAP and ABA. The results are discussedin relation to the ionic changes in the pulvinar motor cells,regulating leaflet movements. Key words: Abscisic acid, auxins, cytokinins, gibberellic acid, pulvinar movements     

INVESTIGATIONS ON AUXIN-GIBBERELLIN INTERACTION ON THE PHOTOTROPISM OF HELIANTHUS ANNUUS HYPOCOTYL

On applying IAA-GB and IAA-NAA mixtures to the hypocotyls ofHelianthus annuus, the plants showed an increase and a decrease,respectively, in their phototropic response compared with thecontrol treated with IAA-H₂O only. In the in vitro experimentsit was observed that the illumination (with 1,500 lux light,lasting for 24 hr) caused photoinactivation of IAA in the mixturesof IAA-H₂O, IAA-GB and IAA-NAA by 50%, 24% and 0%, respectively.In the presence of 0.01% riboflavin as a photosensitizer inthe same mixtures, the degree of photodestruction of IAA causedby the same illumination was 92%, 97% and 86%, respectively. The stronger positive phototropism in the IAA-GB treated hypocotylwas accounted for by the situation that on the illuminated sidethe photo-inactivation of IAA was accelerated by GB, while onthe shaded side IAA and GB acted synergistically. On the otherhand, the weaker phototropism in the IAA-NAA treated hypocotylwas explained as due to a partial inhibition of photoinactivationof IAA by NAA. (Received October 6, 1962; )     

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.     

The Regulation of Invertase Activity in the Latex of Hevea brasiliensis Muell. Arg: THE EFFECTS OF GROWTH REGULATORS, BARK WOUNDING, AND LATEX TAPPING

Treatments which increase latex yield, e. g. bark scraping,latex tapping, and bark application of 2, 4-D or 2-chloroethylphosphonicacid (CEPA) were found to enhance the activity of latex invertase.In previously untapped trees, both the introduction of tappingand the application of 2, 4-D brought about an increase in thelevel of invertase. In regularly tapped trees, the amount oflatex invertase is several times higher than in untapped treesand evidence was obtained that its activity is regulated bythe variation of latex pH. The pH of latex of the clone investigated(PR 107) was shown to vary between 6.3 and 7.1 whereas the activityof invertase, as assayed directly in the latex, has a sharpoptimum at pH 7.5 and falls rapidly with the shift of pH tothe acid side. There was no increase in the content of latexinvertase when trees adapted to regular tapping were treatedwith 2, 4-D. The effect of auxin on actual invertase activitywas essentially mediated through related increase of latex pH.The CEPA and bark scraping were also shown to increase latexpH in tapped trees. The treatment of the bark of tapped trees with CEPA increasedthe level of latex sucrose, as did auxins. Bark scraping alsohad a slight stimulatory effect. The K_m of latex invertase asa function of pH was found to change in the same way as V_max,being highest at pH optimum.     

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.     

High Ambient Carbon-Dioxide does not Affect Respiration by Suppressing the Alternative, Cyanide-resistant Pathway

Total dark respiration (v_t), cytochrome pathway (v_eyt), alternativepathway (v_alt) and residual (v_res) respiration were measuredin Lemna gibba plants, by the use of pathway inhibitors. NaCNwas used to inhibit v_eyt and SHAM (salicylhydroxamic acid) toinhibit v_alt. Residual respiration (v_res) was about 5% of v_t.The effect of high (100 Pa) and low (0 Pa) carbon dioxide partialpressure ([CO₂]) on v_t, v_eyt and v_alt was determined from bothCO₂ efflux and O₂ uptake measurements. The higher [CO₂] suppressedv_t by about 30%. When respiration operated through the cytochromepathway only, in the absence of v_alt, it was suppressed by about12% as measured by the O₂ uptake of submerged Lemna fronds orby about 40% as measured by CO₂ efflux from floating fronds.The higher [CO₂] treatment had only a small effect on respiration,when v_alt alone operated. There was no evidence of a specificsuppression of the v_alt pathway by high [CO₂]. Succinic dehydrogenaseactivity of the mitochondria of roots of Medicago sativum wasreduced by 18%, when the mitochondria were pre-treated with120 as compared to 34 Pa [CO₂]. There was no such effect oncytochrome c oxidase activity of mitochondria under the sameconditions. It is concluded that there is no evidence for the hypothesisthat the high [CO₂] suppression of respiration is a result ofa CO₂ effect on the non-phosphorylating alternative respirationpathway.Copyright 1995, 1999 Academic Press Lemna gibba, Medicago sativum, respiration, cytochrome pathway, alternative pathway, cyanide-insensitive pathway, carbon dioxide, succinic dehydrogenase, cytochrome c oxidase     

The Isolation and Characterization of a Cytochrome b6f Complex from the Cyanobacterium Spirulina sp.

A cytochrome b₆f complex was isolated and purified from Spirulinasp. The complex was solubilized with n-heptyl ß-D-thioglucosideand chromatographed on a DEAE-Toyopearl 650M column. The purifiedcomplex contained a small amount of chlorophyll and carotenoid.At least four polypeptides were present in the complex: cytochromef (29 kDa), cytochrome b₆(23 kDa), iron-sulfur protein (ISP,23 kDa), and a 17 kDa polypeptide. Each polypeptide was separatedfrom the complex treated with 2-mercaptoethanol or urea. Theabsorption spectra of cytochrome b₆ and cytochrome f were similarto those of Anabaena and spinach as expected. The complex wasactive in supporting ubiquinol-cytochrome c oxidoreductase activity.Fifty percent inhibition of the activity was accomplished by1 µM dibromothymoquinone (DBMIB). The K_m values for ubiquinol-2and cytochrome c (horse heart) were 5.7 µM and 7.4 µM,respectively. (Received August 15, 1988; Accepted November 14, 1988)     

The Purification and Characterization of Soluble Acid Invertase from Coleoptiles of Wheat (Triticum aestivum L. cv. Avalon)

Soluble acid invertase from wheat coleoptiles was purified toelectrophoretic homogeneity. A comparison of molecular weightby SDS-PAGE and gel filtration suggested that the enzyme wasa monomer of M_r50 000. The enzyme was a glycoprotein and, afterchemical deglycosylation, possessed a M_rof 48000. A polyclonalantiserum was raised against the deglycosylated protein. Thiscross-reacted specifically with acid invertase. A putative precursorof invertase synthesized in a cell-free translation system wasdetected by SDS-PAGE and fluorography of the immunoprecipitatedpolypeptides. The distribution of acid invertase in wheat seedlingshoots was investigated both by visualizing invertase activityafter starch gel electrophoresis and by immunoblotting. Bothtechniques identified two forms of invertase in extracts ofthe primary leaf and only one form in extracts of coleoptiles.The low pH optimum and the glycoprotein nature of wheat coleoptileinvertase are consistent with a vacuolar location. Fructoseinhibited its activity, suggesting that enzyme activity couldbe modulated by end-product inhibition. Key words: Acid invertase, purification, antiserum, glycoprotein, Triticum aestivum, wheat, coleoptiles     

The Effect of Gibberellic Acid on Starch Breakdown in Sprouting Tubers of Solanum tuberosum L.

The treatment of potato tubers with 150 µmol dm^–3gibberellic acid (GA₃) stimulated starch breakdown and hexoseaccumulation in tuber tissues and the transfer of dry matterto stems. These effects could not be accounted for by enhancedactivities of starch phosphorylase, amylase and acid invertase.Indeed enzyme activities either declined or remained relativelyconstant as starch degradation and hexose accumulation proceeded.Changes in the rate of starch depletion were related to changesin sink strength and sink type, the onset of tuber initiationin controls causing the rate of starch degradation to exceedthat in GA₃-treated tissues, in which tuberization was inhibited. Solanum tuberosum L., gibberellic acid, starch breakdown     

Invertase Activity, Carbohydrate Metabolism and Cell Expansion in the Stem of Phaseolus vulgaris L.

In the stem of Phaseolus vulgaris L. the specific activity ofacid invertase was highest in the most rapidly elongating internode.Activity of the enzyme was very low in internodes which hadcompleted their elongation, in young internodes before the onsetof rapid elongation, and in the apical bud. From shortly afterits emergence from the apical bud the elongation of internode3 was attributable mainly to cell expansion. Total and specificactivities of acid invertase in this internode rose to a maximumat the time of most rapid elongation and then declined. Transferof plants to complete darkness, or treatment of plants withgibberellic acid (GA₃), increased the rate of internode elongationand final internode length by stimulating cell expansion. Bothtreatments rapidly increased the total and specific activitiesof acid invertase in the responding internodes; peak activitiesof the enzyme occurred at the time of most rapid cell expansion. In light-grown plants, including those treated with GA₃, rapidcell and internode elongation and high specific activities ofacid invertase were associated with high concentrations of hexosesugar and low concentrations of sucrose. As cell growth ratesand invertase activities declined, the concentration of hexosefell and that of sucrose rose. In plants transferred to darkness,stimulated cell elongation was accompanied by a rapid decreasein hexose concentration and the disappearance of sucrose, indicatingrapid utilization of hexose. No sucrose was detected in theapical tissues of light-grown plants. The results are discussed in relation to the role of acid invertasein the provision of carbon substrates for cell growth. Key words: Cell expansion, Acid invertase, Hexose, Sucrose, Phaseolus     

The Role of Benzoic Acid and Plant Hormones in Flowering of Lemna gibba G3

The level of benzoic acid was measured in Lemna gibba G3 grownon M and E media under inductive and non-inductive daylengths.Benzoic acid was slightly higher in plants grown on M mediumbut there was no difference in the benzoic acid levels in floweringand vegetative plants. When L. gibba G3 was grown under continuouslight on 1/10 M medium or 1/2 H medium there was virtually noflowering, but addition of benzoic acid to either medium ledto a substantial flowering response. In both cases this floweringresponse was inhibited by the plant hormones IAA, GA₃, ABA andzeatin, with IAA and GA₃ being the least inhibitory and ABAbeing the most inhibitory. This same pattern of inhibition wasseen when L. gibba G3 was grown on M medium under continuouslight, conditions that lead to photoinduction of flowering.These results leave open the possibility that endogenous benzoicacid may interact with other factors to influence the floweringresponse in L. gibba G3. (Received November 13, 1984; Accepted February 27, 1985)     

Control of the Co2 Responses of Stomata by Indol-3ylacetic Acid and Abscisic Acid

Previous studies of stomatal behaviour on detached epidermisof Commelina communis L. have suggested that abscisic acid (ABA)and C02 act independently to cause stomatal closure. Evidenceis presented here that if indol-3ylacetic acid (IAA) is addedto the medium used for incubating the epidermis, an interactionbetween ABA and Co₂ becomes apparent. Increasing concentrationsof IAA reduce the ability of the stomata to respond to CO₂,and ABA appears to antagonize this effect. Recognition of therole of IAA enables us to reconcile earlier conflicting reportsconcerning the interdependence of effects of ABA and Co₂on stomata.     

A Model for the Interaction of Low Temperature, ABA, IAA, and CO2 in the Control of Stomatal Behaviour

In the chilling sensitive (C.S.) species Phaseolus vulgarisit was found that at 22 ?C ABA induced stomatal closure butthis effect was dependent on the presence of CO₂. In the absenceof CO₂ the effect of ABA was completely lost. In contrast toABA, the effect of IAA at 22 ?C was to increase stomatal openingas the IAA concentration increased from 10^–2 to 10 molm^–3, and this effect was dependent upon the presence ofCO₂. However, at 5 ?C the action of ABA was reversed and itwas found to induce stomatal opening when fed via the transpirationstream in excised leaves. Similarly, the CO₂ response characteristicswere reversed at low temperatures as removal of CO₂ from theatmosphere caused stomatal closure. However, the effect of IAAat 5 ?C in the presence of CO₂ and with or without ABA was toincrease stomatal aperture with increasing IAA concentration.Significantly, ABA was found to have no effect upon aperturein the presence of CO₂ when IAA was added. The interactive effectsof ABA, IAA, CO₂ and low temperature are discussed in relationto a model proposed by the authors. Key words: IAA, ABA, CO₂, Stomata     

Gibberellin-colchicine interaction in elongation of azuki bean epicotyl sections

In azuki bean (Azukia angularis = Vigna angularis) epicotylsections, gibberellin A₃ (GA₃) enhanced the growth promotingeffect of indoleacetic acid (IAA), but showed no growth effectwhen applied alone. Sections showed practically no cell division.The promoting effect of GA₃ on section growth seems to be dueto its promoting effect on cell elongation. The diameters of sections treated with IAA increased, but thediameters of sections treated with GA₃ together with IAA remainedconstant. GA₃ seems to suppress cell expansion in a directiontransverse to the cell axis. Colchicine at a concentration with no inhibiting effect on IAA-inducedelongation almost completely reversed the effect of GA₃ On the basis of these results, the participation of wall microtubulesin GA₃-induced elongation is discussed. (Received October 22, 1971; )     

Studies on cytochromes in photosynthetic electron transport system I. photoreduction and photo-oxidation of cytochrome b559 by photosystem II in spinach chloroplasts

Light-induced redox-reactions of cytochrome b₅₅₉ in spinachchloroplasts were investigated. Illumination of chloroplastsinduced photoreduction of cytochrorne b₅₅₉ Red light (650 nm)was more effective than far-red light (725 nm), indicating thatthe photoreduction is a photosystem II-mediated reaction. Onaddition of DCMU, the photoreduction was eliminated and a photooxidationof cytochrome b₅₅₉ was observed. The rate of this photooxidationwas faster with photosystem II light than with photo-systemI light. On addition of Mn⁺⁺ the photooxidation was partly suppressed;far-red light became as effective as red light in inducing photooxidationof cytochrome b₅₉₉, in the presence of DCMU and Mn⁺⁺. Ascorbate completely suppressed photooxidation of cytochromeb⁵⁵⁹ In the presence of ascorbate, however, photooxidation wasobserved in the presence of inhibitors or after inhibitory treatmentsof chloroplasts which affected the oxidizing side of systemII. These inhibitors and inhibitory treatments, but not DCMU,decreased the redoxpotential of cytochrome b₅₅₉. Reactivationof Hill reaction in Tris-washed chloroplasts by indophenol-ascorbatetreatment was not accompanied by an abolishment of photooxidationof cytochrome b₅₅₉. A possible mechanism is proposed to account for these reactionsof cytochrome b₅₅₉ in the photosynthetic electron transportin chloroplasts. (Received April 4, 1972; )     

Interaction of a Brassinosteroid with IAA and GA3 in the Elongation of Cucumber Hypocotyl Sections

A synthetic brassinosteroid, 22,23(S,S)-homobrassinolide (hBR),was examined for its interaction with IAA and GA₃ in the elongationof hypocotyl sections of light-grown cucumber (Cucumis salivusL. cv. Aonagajibai) seedlings. hBR alone was less active thanIAA. Its optimal concentration was around 10 µM and thelowest effective concentration between 10 and 100 µM,which is more than 100 times higher than that of brassinolide.hBR was more active in sections from younger seedlings. Itsgrowth-promoting effect was negated or greatly reduced by inhibitorsof auxin-induced elongation such as p-chlorophenoxyisobutyricacid and kinetin. hBR acted synergistically with IAA and 2,4-Dbut not with GA₃ showing only an additive effect. Sequentialtreatment of sections with hBR and then with IAA also resultedin synergistic enhancement of auxininduced elongation, but whenthe order of treatment was reversed, hBR was inactive. The synergisticeffect was obtained with 1 h pretreatment with hBR and couldbe reduced by subsequent washing with water. There was no sequentialinteraction between hBR and GA₃. The synergistic pretreatmenteffects of hBR and GA₃ were simply additive to each other. Amembrane-bound ATPase inhibitor, dicyclohexylcarbodiimide, inhibitedthe hBR-induced elongation, but did not affect GA₃-induced elongation.The findings led to the conclusion that brassinosteroids enhanceauxin action and possess growth-promoting activity which isindependent of that of gibberellin. (Received November 9, 1984; Accepted February 18, 1985)