In Vitro Phosphorylation of Proteins in IAA-Treated Mesocotyls in Zea mays

Five-mm sections of elongation zones of Zea mesocotyls wereincubated for designated periods with various concentrationsof IAA. In vitro protein phosphorylation in the soluble fraction(85,000 x g supernatant) prepared from the sections was analyzedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The phosphorylation of proteins in the soluble fraction thathad been prepared from sections incubated for 20 min in thepresence of 10{small tilde}^s M IAA was greater than that inthe sections incubated for 20 min without IAA. The amount ofphosphorylation of proteins per protein became higher when higherconcentrations increased (10{small tilde}⁸—10{small tilde}⁵M).The growth of sections incubated in the presence of 10{smalltilde}⁸ M IAA or higher concentrations was greater than thatof sections incubated in the absence of IAA. The promotion ofgrowth by IAA was greater at higher concentrations of IAA. Proteinsin the soluble fraction, prepared from sections incubated for20 min in the presence of 10{small tilde}⁵ M IAA, were phosphorylatedin the presence of either 10 fM cAMP, 10 µM cGMP, 100µM W-7, 100 µM W-5, 20 µM H-7 or 20 µMHA1004. The calmodulin antagonist, W-7, and the inhibitor ofprotein kinase C, H-7, inhibited the phosphorylation of proteinsstimulated by incubation with IAA. These results suggest thatIAA promotes cell elongation via protein phosphorylation thatdepends on calmodulin-dependent protein kinase and protein kinaseC. (Received November 29, 1995; Accepted May 20, 1996)     

Endogenous Hormones in Seeds, Germination Behaviour and Early Seedling Characteristics in a Normal and ogura Cytoplasmic Male Sterile Line of Rapeseed (Brassica napus L.)

Endogenous hormones, namely cytokinins (CKs), indole-3-aceticacid (IAA) and abscisic acid (ABA) were quantified by specificenzyme-linked immunosorbent assay (ELISA) in the mature seedof normal (cv. Westar) and ogura (ogu) cytoplasmic male sterile(CMS) lines of rapeseed (Brassica napus L.). Dihydrozeatin (DZ)and dihydrozeatin riboside (DZR) were the major CK base andriboside, respectively, in seeds of both the normal and oguCMS lines. The normal seed had more than 4-fold DZ levels incomparison to that of ogu CMS. On the other hand, the ogu CMSseed had higher levels of CK o-glucosides and CK. nucleotidesthan normal seed. Seeds of the normal line contained 5-foldmore IAA but had one-quarter the level of ABA in comparisonto those of the ogu CMS line. The normal line also had greaterseed diameter and weight than the ogu CMS line and the normalseed germinated earlier than the male sterile seed. DZ (10^–6M) promoted the germination of ogu CMS seeds, but it was notcomparable to that of the normal line. ABA (10^–6 M) inhibitedseed germination of ogu CMS but had little effect on the normalline. The normal seedlings had shorter primary roots, more lateralroots, longer hypocotyls, greater cotyledon fresh weight andhigher chlorophyll levels in comparison to ogu CMS seedlings.Exogenously supplied DZ, IAA and ABA affected the various parametersof both the normal and ogu CMS seedlings, but in most casesdid not fully restore the differences in the two lines. The results presented show that in the ogura cytoplasmic malesterile line of B. napus (1) a number of seed and seedling characteristicsare affected, and (2) the altered seed morphology is accompaniedby changes in the levels of various hormones. Key words: Brassica napus, cytoplasmic male sterility, enzyme-linked immunosorbent assay, hormone, seed germination     

Changes in Endogenous Ribosyl-trans-zeatin and IAA Levels in Relation to the Proliferation of Tobacco Crown Gall Cells

Changes in the contents of major endogenous plant hormones intobacco crown gall cells, namely IAA and ribosyl-trans-zeatin,during cell growth were examined using HPLC and ¹⁴C-labeledplant hormones. The content of IAA was high at the early logarithmicstage, while that of ribosyl-trans-zeatin was high at the middlelogarithmic stage. This suggests that cell growth is affectedfirst by IAA, then by ribosyl-trans-zeatin. ³ Present address: Department of Agricultural Chemistry, TottoriUniversity, Koyama, Tottori 680, Japan (Received July 13, 1981; Accepted September 11, 1981)     

Effect of Indoleacetic Acid on Growth and Dinitrogen Fixation in Cyanobacteria

The effect of IAA on growth, dinitrogen fixation, and heterocystsfrequency of Anabaena PCC 7119 and Nodularia sp. have been investigated.Concentrations of IAA ranging from 10^–10 to 10^–4M did not change the growth of Anabaena PCC 7119. Concentrationshigher than 10^–4 M were inhibitory. Similar results werefound in Nodularia sp. although in this case the inhibitoryeffect appeared with 10^–5M of IAA. Neither the nitrogenaseactivity nor the heterocysts frequency were enhanced by IAAtreatment. (Received June 17, 1986; Accepted January 22, 1987)     

Resemblance of growth substances to metal chelators with respect to their actions on duckweed growth

Action patterns of IAA, KIN and GA on the growth of Lemna gibbaG3 are similar to those reported for agents chelating both cupricand ferric ions. Relatively high doses of growth substances,e.g.10^–6 M IAA or KIN and ^–4 M GA, inhibit developmentof photoperiodically induced flower buds and antagonisticallypromote frond multiplication; whereas, at relatively low doses,e.g. 10^–9 M IAA or KIN and ^–5 M GA, they acceleratethe process occurring in the latter half of the induction periodto enhance flower induction. Complex forming abilities of IAA,KIN and GA with cupric and ferric ions are demonstrated spectrophotometrically.Moreover, the ferrous ion-dependent oscillatory change in reproductiveand vegetative photophilies of duckweed is eliminated by KINbut not by IAA and GA. Of the three growth substances tried,KIN alone shows an affinity for ferrous ions. (Received April 5, 1971; )     

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; )     

Epidermal Growth Factor Interacts with Indole-3-Acetic Acid and Promotes Coleoptile Growth

We used coleoptile sections of Avena sativa, Sorghum bicolor,and Zea mays seedlings to examine interactions between epidermalgrowth factor (EGF) and indole-3-acetic acid (IAA) that mayaffect plant growth and development. Our 24-h bioassays employedthree controls ranging in dilution from 10^–4 to 10^–8g ml^–1: (1) 50 mM potassium-phosphate buffer solution(pH=6.0), (2) bovine serum albumin, a nonspecific protein; and(3) IAA; plus two treatments: (1) mouse epidermal growth factor(EGF) ranging from 10^–6 to 10^–10gml^–1, and(2) EGF + IAA. In all three species growth in IAA, EGF, andEGF + IAA treatments showed significant increases over controls;EGF+IAA showed significant increases in growth over IAA alone.As the concentrations of IAA decreased, the EGF and IAA interactionbecame more pronounced. At the highest IAA concentrations, EGF+ IAA increased growth rates ca. 2% to 39%, whereas at lowerIAA concentrations EGF + IAA promoted growth as much as 121%,thereby lowering the normal IAA physiological set point up tothree or four orders of magnitude. Our data suggest that aninteraction between EGF and IAA may allow plants to recognizeand respond to animal biochemical messengers, resulting in changesin plant cell elongation that ultimately may alter plant growthpatterns. (Received April 27, 1994; Accepted September 5, 1994)     

Effects of Osmotic Stress, Ionic Stress and IAA on the Cell-Membrane Resistance of Vigna Hypocotyls

The cell-membrane resistance (R_m) of Vigna hypocotyls was examined,and the effects of osmotic stress, ionic stress and IAA on R_mwere investigated. R_m decreased by 64 to 77% under osmotic stressin the presence of absorbable solutes (40 mM sorbitol, 15 mMKC1, 30 mM sucrose; or 40 mM sorbitol, 15 mM KC1, 30 mM sucroseplus 10^–4 M IAA) or under ionic stress (50 mM NaCl or50 mM KC1). R_m was not changed by perfusion with 10^–4M IAA. Therefore, the hyper-polarizations of the membrane potentialobserved in both cases should be ascribed totally to the activationof the electrogenic proton pump. Although R_m showed an increaseof 1.6 fold when the hypocotyls were subjected to osmotic stress(100 mM sorbitol or 100 mM sorbitol plus 10^–4 M IAA),83.6% or 92.4% of the hyperpolarization of the membrane potential(V_px was also the result of the activation of the pump. Theresults, calculated on the basis of the current source model,support the viewpoint that the hyperpolarization of the cellmembrane potential of Vigna hypocotyls under osmotic stress,ionic stress or in the presence of IAA is an expression of theactivation of the proton pump, and is not caused by an increasein R_m. ¹ Present address: Researchers and Planners of Natural Environment, Yotsugi Bldg. (2F), 1-5-4 Horinouchi, Suginami-Ku, Tokyo,166 Japan ² Present address: Graduate School of Integrated Science, YokohamaCity University, 22-2 Seto, Kanazawa-Ku, Yokohama, 236 Japan (Received February 14, 1991; Accepted July 24, 1991)     

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; )     

Further Studies of Electro-Osmosis in Nitella in Relation to Pores in Membranes

By measuring the number of water molecules per ion which weremoved electro-osmotically through cells of Nitella translucensand N. flexilis it has been shown that a significant differenceexisted between samples of these species in 1965. In each speciesthe electro-osmotic efficiency was greater with Na⁺ than withK⁺⁺. Also 10^-4 to 10^-5 M IAA tended to decrease electro-osmoticefficiencies while IAA, after 30 min. treatment, produced asignificant increase in water flow into the treated end of aliving cell. Calculations based on this work suggest that about10⁸–10⁹ pore sites per cm² exist on the surface membranesfor Na⁺ or K⁺ ion transport.     

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by Agrobacterium tumefaciens III. Formation of IAA Conjugates in Cultured Carrot Tissues Transformed with Wild-type and Mutant Ti Plasmids

Inactivation of IAA was examined in cultured carrot tissuestransformed with Agrobacterium tumefaciens that harbored wild-type,aux^– or cyt^– Ti plasmids. IAA oxidase activities were similar among the three types oftransformed tissue. Metabolites of IAA were examined by followingthe fate of 3-indolyl-[l-^l4C]IAA. IAA conjugates were detectedin all transformed tissues as well as in hypocotyl segmentsof non-transformed carrot seedlings. The rate of formation ofIAA conjugates was ten times higher in the tissues transformedwith wild-type or cyt^– Ti plasmids than in the tissuestransformed with aux^– Ti plasmids. When the tissues transformedwith aux^– Ti plasmids were cultured on medium that containedIAA for 6 h, the rate of formation of IAA conjugates in thesetissues became as high as that in tissues transformed with wild-typeor cyt^– Ti plasmids. The tissues transformed with wild-type or cyt^– Ti plasmidsnot only synthesize larger amounts of IAA but also convert alarger amount of free IAA to conjugated IAA than do non-transformedand aux^– transformed tissues. Presumably, in carrot, theformation of IAA conjugates decreases the amount of free IAAin the transformed tissues that synthesize large amounts ofIAA and, consequently, the level of free IAA can be maintainedfairly constant. (Received June 2, 1989; Accepted May 23, 1990)     

Effects of Inhibitors of Protein Synthesis on Transmembrane Auxin Transport in Cucurbita pepo L. Hypocotyl Segments

Pretreatment of 2?0 mm segments of etiolated zucchini (Cucurbitapepo L.) hypocotyl with cycloheximide (CH) or 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide(MDMP) eliminated the stimulation by N-1-naphthylphthalamicacid (NPA) of net uptake of [1-¹⁴C]indol-3yl-acetic acid ([1-¹⁴C]IAA),but had relatively little effect on the net uptake of IAA inthe absence of NPA. The efflux of [1-¹⁴C]IAA from preloadedsegments was not substantially affected by inhibitor pretreatmentin the absence of NPA, but CH pretreatment significantly inhibitedthe reduction of efflux caused by NPA. Pretreatment with CHor MDMP did not affect net uptake by segments of the pH probe[2-¹⁴C]5,5-dimethyl-oxazolidine-2,4-dione ([2-¹⁴C]DMO), or thenet uptake of [¹⁴C]-labelled 3-O-methylglucose ([¹⁴C]3-0-MeGlu),suggesting that neither inhibitor affected intracellular pHor the general function of proton symporters in the plasma membrane.Both compounds reduced the incorporation of label from [³⁵S]methionineinto trichloroacetic acid (TCA)-insoluble fractions of zucchinitissue, confirming their inhibitory effect on protein synthesis. The steady-state association of [³H]IAA with microsomal vesiclesprepared from zucchini hypocotyl tissue was enhanced by theinclusion of NPA in the uptake medium. The stimulation by NPAof [³H]IAA association with microsomes was substantially reducedwhen the tissue was pretreated with CH. However, CH pretreatmentdid not affect the level of high affinity NPA binding to themembranes indicating that treatments did not result in lossof NPA receptors. It is suggested that the auxin transport site on the effluxcarrier system and the receptor site for NPA may reside on separateproteins linked by a third, rapidly turned-over, transducingprotein. Key words: Auxin carriers, auxin efflux, Cucurbita pepo, phytotropin receptors     

Hormonal Control of Xylogenesis in Pith Parenchyma Explants of Lactuca

The time course of xylem differentiation was determined in explantsof lettuce pith parenchyma (Lactuca sativa L. cv. Romana) culturedon Murashige and Skoog (1962) medium using different concentrationsof auxin (IAA) and one cytokinin (zeatin or kinetin). Increasinglevels of auxin from I mg 1^–1 to 15 mg 1^–1 in thepresence of a constant level of a cytokinin (zeatin or kinetin)yielded up to 10 mg 1^–1 IAA, an increase in the numberof tracheary element formations. Cytokinin concentrations aboveand below o.1 mg 1^–1 interacting with an optimal xylogenicamount of auxin inhibited xylogenesis. The IAA (10 mg 1^–1)-zeatin(0.1 mg ^1–1) treatment produced the greatest number oftracheids, while kinetin compared to zeatin did not producesuch an effect. The different effectiveness of zeatin and kinetinin inducing tracheary element formations was not due to a differentcapacity of the two cytokinins to stimulate cell division butit seems likely that zeatin, because of interaction with IAA,is more active than kinetin in the determination of the dividingcells in a specific type of cytodifferentiation. The IAA (10mg 1^–1)-zeatin (0.1 mg 1^–1) treatment produced about6.9 per cent tracheids with respect to cell division while IAA(10 mg 1^–1)-kinetin (0.1 mg 1^–1) produced 4.2 percent. These results are discussed with reference to the problemsof hormonal control of xylem differentiation.     

B-Nine Reversal of Benzimidazole-or IAA-induced Inhibition of Growth and Yield of Cowpea Plants

Growth, flowering, and yield of Cowpea (Vigna catjang) wereinhibited by Benzimidazole (10^-3M) or IAA (10^-2M). These growthinhibitions were reversed by B-Nine (2x10^-3M) which alone waswithout any effect. In combination, IAA and Benzimidazole weremutually antagonistic. A common mechanism is suggested for theB-Nine reversal of the Benzimidazole or IAA inhibition of growth.     

Auxin-promoted Transport of Metabolites in Stems of Phaseolus vulgaris L.: SOME CHARACTERISTICS OF THE EXPERIMENTAL TRANSPORT SYSTEMS

Indol-3yl-acetic acid (IAA) applied to sterns of Phaseolus vulgarisseedlings, decapitated above primary leaves, enhanced the mobilizationof ¹⁴C-metabolites to the treated stumps and this effect wasapparent within 3–6 h of applying the hormone. More than90 per cent of the total ¹⁴C-activity transported to the stumpswas detected in the alcohol-soluble extracts. In all treatments,less than 5 per cent of the ¹⁴C-photosynthate exported fromthe primary leaves was translocated upwards. Accumulation of¹⁴C-activity was also increased when the IAA was applied laterallyto intact internodes. This effect was obtained when ¹⁴C wassupplied either above or below the point of hormone application.By selective heat girdling, it was shown that the auxin affected¹⁴C transport when either the root ‘sink’ was removedor transpiratory flow of water through the treated internodewas maintained. Decapitated stems treated with plain lanolinfor 3 d were found to retain their responsiveness to auxin interms of enhanced metabolite transport. Heat-girdling experimentsand estimates of ¹⁴C transport velocity suggested that mostof the ¹⁴C movement was restricted to the phloem of treatedstumps. Similar effects of IAA on a transport in excised stemsegments of Phaseolus vulgaris were observed.     

Effects of indoleacetic, p-chlorophenoxyisobutyric and 2,4,6-trichlorophenoxyacetic acids on three phases of rooting in Azukia cuttings

In adventitious root formation of disbudded epicotyl cuttingstaken from light-grown, 5-day-old Azukia angularis seedlings,indoleacetic acid (IAA), 1 x 10^–4 M, applied during thefirst day showed no effect, but enhanced the effect of IAA,1 x 10^–4 M, applied during the second day. Treatment duringthe second day promoted rooting by about 70%, and a combinationof treatments for the first and second days promoted rootingsome 200%. p-Chlorophenoxyisobutyric acid (PCIB), 3 x 10^–4M, and2,4,6-trichlorophenoxyacetic acid (2,4,6-T), 2 x 10^–44M, applied the first day also enhanced the effect of IAA, 2x 10^–4 M, applied the second day. When applied the second day, PCIB, 2 x 10^–4M, increasedthe number of root primordia or clusters of small cells, butnot die number of protruded roots. Formation of the cell clusterwas inhibited by 2,4,6-T, 3 x 10^–4M, applied the secondday. Rooting processes in Azukia cuttings seem to include at leastthree phases: the first phase is induced not only by IAA butalso by PCIB or 2,4,6-T, the second phase is induced by IAAor PCIB and the diird phase depends specifically on IAA. (Received October 28, 1970; )     

The Polarity of Movement of Endogenously Produced IAA in Relation to a Gravity Perception Mechanism

The polarity of radioactive IAA transport in segments of theleaf sheath base of Avena fatua L. is reversed upon their inversionthrough 180° transport towards the apex being greater thantowards the base. Changes in rates of transport leading to thisreversal can be detected within 10–20 min, correlatingwith the timing of statolith movements from the base to theapex of statocyte cells and conforming to a proposed model forthe gravity control of auxin transport. Transport of H³-trytophan-derived H³-IAA was found to undergosimilar changes in polarity upon re-orientation as did exogenouslyapplied H³ or C¹⁴ IAA. It is concluded that the proposed modelalso relates to the movement of endogenously produced IAA.     

Stomatal responses of Vicia faba L. to indole acetic acid and abscisic acid

Evidence is presented that stomata in isolated epidermal peelsof Vicia faba L. open in darkness in response to the externalpresence of indole acetic acid (IAA) in the incubation medium.The effect of IAA is found to be overcome completely in thepresence of either TRIS or MES buffers. In the absence of buffer,V. faba stomata are shown to be influenced by IAA in a concentration-dependenttrend which reached a maximum at an [IAA] of 10^–3 molm^–3. Further investigations reveal that stomata in thisspecies can be shown to respond to the presence of IAA and anotherphytohormone, abscisic acid (ABA). IAA and ABA are demonstratedto be antagonistic in their effects provided the incubationconditions are suitable. The data are discussed in relationto stomatal responses of other species in different treatmentconditions. Recommendations are made with respect to standardizationof incubation media during epidermal peel experiments. Key words: Vicia faba, stomata, indole acetic acid, abscisic acid, buffers