Adventitious root formation 'in vitro' in apple rootstocks (Malus pumila) II. Uptake and distribution of indol-3yl-acetic acid during the auxin-sensitive phase in M.9 and M.26

During the auxin-sensitive phase of root initiation, rates of 3-indolyl- [2-¹⁴C] acetic acid (IAA) uptake into the 1 cm bases of shoots of the apple rootstock M.9 ( Malus pumila Mill.) 'in vitro' were not significantly affected by the presence of 10⁻³ M phloroglucinol (PG) using either liquid or agar-solidified media. The use of a liquid medium did however reduce rates of uptake over a 10-day period of auxin application. The distribution of labelled IAA between the 1-cm base and the shoot remainder was not affected by PG.
Exposure of shoots of the difficult-to-root M.9 and the easy-to-root M.26, to 2.8 × 10⁻⁵ M IAA containing [2-¹⁴C] IAA revealed no positive correlation between the amount of label taken up by the 1-cm base and rooting performance. M.9 bases absorbed almost twice as much label as M.26 after 9 days but had produced only one-third as many roots. Measurements of label distribution between the 1-cm base and the shoot remainder showed that less than 10% of the label moved to the shoot remainder over a 6-day period of auxin application. Dose-response curves of IAA and rooting over the range 1 × 10⁻⁵ M and 3 × 10⁻³ M showed that root number in M.9 was at an optimum at 1 × 10⁻³ M IAA after 6 days whilst M.26 required only 1 × 10⁻⁴ M for a similar response. These data support the hypothesis that differences in rooting of the two rootstocks reflect differences in the endogenous metabolism of exogenous IAA and not differences in its rates of uptake or distribution in the shoots.     

Effect of auxins on spermidine uptake into carrot protoplasts

The effect of an auxin, indole-3-acetic-acid (IAA), on spermidine uptake into protoplasts of carrot ( Daucus carota L. cv. Ingrid) was studied. In the presence of 1 m M Ca²⁺, IAA (10⁻⁷ to 10⁻⁴ M ) enchances [¹⁴C]-spermidine uptake into carrot protoplasts, while no stimulation occurs in the absence of Ca²⁺. The time course of the uptake with and without IAA is very rapid and reaches saturation within 1 to 2 min. Preincubation of protoplasts with IAA inhibits the spermidine uptake. La³⁺, known not to penetrate the plasmalemma, exerts the same effect as Ca²⁺, but gives lower uptake values than Ca²⁺. The application of vanadate, an ATPase inhibitor, strongly inhibits IAA-stimulated spermidine uptake, suggesting that an energy-dependent mechanism may be involved in this transport. Neither spermidine nor Ca²⁺ alone stimulate IAA uptake. The synthetic auxin 2,4-dichlorophenoxyacetic acid, yields the same results as IAA with regard to time course of spermidine uptake with and without preincubation while, unlike IAA, no significant effect was observed on the Ca²⁺ -induced increase of spermidine uptake.     

Vessel differentiation along different tissue polarities

Organized vessel differentiation in an isolated system was studied on a quantitative basis. An auxin source was oriented on isolated pieces of turnip storage root ( Brassica campestris cv. Rapifera) to allow diffusion a) in the direction of, b) at right angle to, and c) opposite to the original polarity. New vessel members differentiated within 44 h, and a minimum average auxin (IAA) concentration of 3.10⁻⁶ M was required to induce initial vessel differentiation. The differentiation rates in three experimental orientations were 167, 60 and 43 cells h⁻¹ at 10⁻³ M IAA, and 1445, 1346 and 838 cells (log IAA concentration)⁻¹ after 96 h, respectively. The difference between the differentiation rates in the original polarity orientation (a) and in the orientation at right angle (b) is interpreted as reflecting reorientation itself, which requires a minimum time.     

The Relationship of Indole-3-Acetic Acid Content and Growth of Crown-Gall Tumor Tissues of Tobacco in Culture

We have measured the content of the auxin, indole-3-acetic acid (IAA), in a cloned, crown-gall teratoma line of Nicotiana tabacum L. cv. 'Turkish' by a highly specific and sensitive radioimmunoassay. This tissue line, which does not require auxin for continuous growth in culture, exhibits two phases of growth. During the first phase, which lasts about two weeks after subculturing, growth is exponential on a fresh weight basis and the content of IAA is about 10⁻⁷–10⁻⁶ mol. kg⁻¹, but variable. During the second phase, growth rate declines gradually and the IAA content of the tissue drops dramatically; however, this drop does not result from a net loss of auxin by the tissue. The rate of growth during the exponential phase was not correlated with the IAA content of the tissue, but was strongly correlated with the IAA content of the inoculum. We found that rapidly growing leaves of Turkish tobacco have roughly the same IAA content as cultured teratoma tissues. Moreover, both tissues exhibited a similar relationship between auxin content expressed per leaf or per tissue explant and growth. These findings do not support the hypothesis that the autonomous growth of plant tumors results from an abnormally high content of auxin in the tissue.     

Flooding resistance of Rumex species strongly depends on their response to ethylene: Rapid shoot elongation or foliar senescence

In excised Zea maus L. coleoptiles incubated in aerated media at high fresh weight per volume ratios, indole-3-acetic acid induces transient drops of extracellular pH. Based on the quantitative dependency of the response on the initial auxin concentration we developed a novel auxin bioassay, which allows reliable estimation of IAA concentrations between 10⁻⁸⁵ and 10⁻⁵M. Using the bioassay and complementary concentration measurements by IAA fluorescence we found the transient IAA-induced pH response paralleled by a decrease of auxin activity and concentration in the medium. This decline is rapid and starts immediately upon auxin addition, and insofar differs from the well known IAA degradation by epiphytic bacteria in long-term auxin tests. We conclude that the transient character of the auxin pH response is due to rapid IAA metabolism. The effect occurs under those experimental conditions that are necessary for reliable estimations of auxin-induced shifts of cell wall pH, which considerably complicates the interpretation of the results.     

Osmotic stress- and indole-3-butyric acid-induced NO generation are partially distinct processes in root growth and development in Pisum sativum

In this work, the effects of osmotic stress and exogenous auxin (indole-3-butyric acid, IBA) on root morphology and nitric oxide (NO) generation in roots were compared in pea plants. Five-day-old plants were treated with 0, 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸ or 10⁻⁹  M IBA or with PEG 6000 at concentrations that determined 0, 50, 100, 200 or 400 mOsm in the medium, during 5 days. NO generation was examined by in situ and in vivo fluorescence method. Increasing concentrations of PEG as well as IBA resulted in shortening of primary root (PR), enhancement of lateral root (LR) number and significant increase of NO generation. Time-dependent investigations revealed that in the case of IBA treatments, the LR number increased in parallel with an intensified NO generation, while elongation of PR was not followed by changes in NO levels. Under osmotic stress, the time curve of NO development was distinct compared with that of IBA-treated roots, because significantly, the appearance of lateral initials was preceded by a transient burst of NO. This early phase of NO generation under osmotic stress was clearly distinguishable from that which accompanied LR initiation. It is concluded that osmotic stress and the presence of exogenous auxin resulted in partly similar root architecture but different time courses of NO synthesis. We suppose that the early phase of NO generation may fulfill a role in the osmotic stress-induced signalization process leading to the modification of root morphology.     

Effect of zeatin on the growth and indolyl-3-acetic acid and abscisic acid levels in maize roots

Elongation, indolyl-3-acetic acid (IAA) and abscisic acid (ABA) levels, – gas chromatography-mass spectrometry quantification –, in the elongating zone were analysed for maize ( Zea mays L., Cv. LG11) roots immersed in buffer solution with or without zeatin (Z). The effect of Z depends on the initial extension rate of roots. The slower growing roots are more strongly inhibited by Z (10⁻⁷−10₋₅ M ) and they show a greater increase in IAA and ABA content. When compared to the rapidly growing roots, the larger reactivity of the 'slow'ones cannot be attributed to a higher Z uptake as shown when using [¹⁴C]-Z. It is suggested that Z could regulate root elongation by acting on the IAA and/or ABA level. The comparative action of these two hormones is discussed.     

Roles of auxin and gibberellic acid in growth and maturation of epicotyls of Vigna angularis: Cell wall changes

The effects of auxin and gibberellic acid on cell wall composition in various regions of epicotyls of azuki bean ( Vigna angularis Ohwi and Ohashi cv. Takara) were investigated with the following results. (1) Young segments excised from apical regions of the epicotyl elongated in response to added 10⁻⁴ M indole-3-acetic acid (IAA). When the segments were supplied with 50 m M sucrose, the IAA-induced segment growth was accompanied by enhanced overall synthesis of cell wall polysaccharides, such as xyloglucans, polyuronides and cellulose. This IAA effect on the cell wall synthesis is a consequence of extension growth induced by IAA. Gibberellic acid (GA) at 10⁻⁴ M synergistically enhanced the IAA-induced cell wall synthesis as well as IAA-induced extension growth, although GA by itself neither stimulated the cell wall synthesis nor extension growth. In the absence of sucrose, cell wall synthesis was not induced by IAA or GA. (2) In mature segments excised from basal regions of the epicotyl, no extension growth was induced by IAA or GA. GA enhanced the synthesis of xylans and cellulose when the segments were supplied with 50 m M sucrose. IAA had no effect on the cell wall synthesis. These findings indicate that synthesis of polyuronides, xyloglucans and cellulose, which occurs during extension growth of the apical region of the epicotyl, is regulated chiefly by auxin whereas synthesis of xylans and cellulose during cell maturation in the basal region of the epicotyl is regulated by GA.     

Vitamin D3 affects growth and Ca2+ uptake by Phaseolus vulgaris roots cultured in vitro

Vitamin D₃ at low concentration (10⁻⁹ M) inhibited the growth of Phaseolus vulgaris L. (cv. Contrancha) roots in vitro as measured by elongation (14 h) and [³H]-leucine incorporation into protein (2 h), and increased their labelling with ⁴⁵Ca²⁺ (2 h). Cycloheximide and puromycin (50 u.M) blocked vitamin D₃ stimulation of root ⁴⁵Ca²⁺ labelling, indicating that it is mediated by de novo protein synthesis. The calcium ionophore X-537A (10⁻⁵JW) induced similar changes both in root elongation and ⁴⁵Ca²⁺ uptake (14 h). This may indicate that the inhibitory effects of the sterol on root growth are mediated by changes in Ca²⁺ fluxes. However, this interpretation should be further strengthened by additional studies as the ionophore may have acted on root growth, affecting physiological processes other than Ca²⁺ transport.     

Effects of a brassinosteroid on growth and electrogenic proton extrusion in maize root segments

In apical or subapical root segments of maize ( Zea mays L. cv. Dekalb XL640), 10⁻⁷–10⁻⁵ M 24-epibrassinolide (BR), a physiologically active synthetic epimer of the pollen hormone brassinolide, induces a significant stimulation of root growth, associated with an increase of acid secretion. The increase in acid secretion is enhanced by the presence of K⁺ in the medium and is accompanied by an early, significant hyperpolarization of the transmembrane electric potential (PD), which is completely suppressed by the addition of the protonophore uncoupler FCCP. Similar effects of BR have earlier been reported for shoots, and also for IAA in shoots. Contrariwise, 10⁻⁸–10⁻⁷ M IAA inhibits acid secretion and depolarizes the PD in the maize root segments. This suggests different pathways for the action of the two different hormones on the proton pump.     

Growth, auxin requirement, and indole-3-acetic acid content of cultured crown-gall and habituated tissues of tobacco

Abstract. We used radioimmunoassay to compare the indole-3-acetic acid (IAA) concentration in cultured auxinautonomous tissues of Nicotiana tabacum L. cv.'Havana 425'that differ in growth rate, auxin requirement, capacity for organogenesis, and proximal cause of transformation. Lines HT37 and HB6 were crown-gall tissues transformed by the T37 and B6 strains of Agrobacterium tumefaciens . The tissue line 156AH, like the crown-gall lines, was auxin and cytokinin autotrophic, but arose spontaneously in culture and did not result from crown-gall transformation. The IAA content of the lines was variable and between 10⁻⁷ and 10⁻⁶ moles kg⁻¹ fresh weight. This same range of IAA concentration was found in growing leaf tissues on the plant. IAA concentrations were 2–5 fold higher in HB6 than HT37 during the first few days of culture, and exogenous auxin induced the teratomatous HT37 tissue to grow in an unorganized fashion suggesting that auxin plays a role in regulating tumor morphology. The major difference between genetically transformed crown-gall tissues and the auxin-cytokinin autotrophic tissue line was that net IAA production continued to rise for several weeks of culture in tumor tissues but only for the first few days of culture in the autotrophic tissue.     

Translocation and utilization of carbon in wheat (Triticum aestivum)

Wheat ( Triticum aestivum L. cv. SUN 9E) was grown in a growth chamber under conditions of low soil nitrogen. Translocation of carbon to the roots and the subsequent utilization of these carbohydrates was determined. In vegetative plants (22 days old), 21.5 mg C day⁻¹ were translocated to the roots. 29% of this was incorporated into dry matter, 32% was respired (28% via the cytochrome and 4% via a SHAM-sensitive, presumably the alternative nonphosphorylating, pathway) and 39% was translocated back to the shoots, mainly in the form of amino acids. – The rote of root maintenance respiration during the vegetative phase was estimated to be 0.7 mg O₂ h⁻¹ (g dry weight of roots)⁻¹ and the root growth respiration to be 0.41 g O₂ (g dry weight of roots)⁻¹. Total carbohydrate utilization due to root respiration via the alternative, nonphosphorylating pathway during the major part of the growth period was calculated to be only ca 6% of carbohydrate utilization for grain growth. The rate of specific mass transfer (SMT) of sugars in the sieve tubes was estimated from the data on C-translocation and data on the total area occupied by sieve tubes in a cross section of the root system. SMT was calculated to be 0.8 mg sucrose s⁻¹ cm⁻², which is very similar to the published value on SMT for other organs, except roots.     

The rooting ability of shoots raised 'in vitro' from the apple rootstock A2 in juvenile and in adult growth phase

The apple rootstock A2 can be readily propagated in vitro both in the juvenile and in the adult growth phase. Shoots were produced by meristem tip culture from the apple rootstock A2 in different growth phases. The influence of growth phases and different concentrations of PG and IBA was investigated as to rooting percentage, survival percentage, number of roots per rooted shoot, root length, shoot length and formation of callus. IBA at 15 μ M without PG gave a significantly lower rooting percentage than 5 and 10 μ M IBA. PG together with IBA stimulated rooting, the optimum concentrations of PG being, however, not the same for the different growth phases. For the adult growth phase, 10⁻⁴ M PG promoted rooting, whereas 10⁻³ M PG markedly inhibited rooting. In the juvenile growth phases, both 10⁻⁴ and 10⁻³ M PG stimulated rooting. PG at 10⁻⁴ M also increased the number of roots. The longest roots were obtained at 10⁻³ M PG and 5 μ M IBA. PG at 10⁻³ M reduced callus formation at all IBA concentrations used. Neither shoot length nor root length influenced the survival percentage.     

Adventitious root formation 'in vitro' in apple rootstocks (Malus pumila) I. Factors affecting the length of the auxin-sensitive phase in M.9

The length of the auxin-sensitive phase of root initiation 'in vitro' in the apple rootstock M.9 ( Malus pumila Mill.) has been determined using the auxins indol-3yl-acettc acid (IAA) at 2.8 × 10⁻⁵ M and indol-3yl-butyric acid (IBA) at 1.5 × 10⁻⁵ M in the presence and absence of 10⁻³ M phloroglucinol (PG). PG synergised IBA-induced rooting after 4 days exposure, but contact times exceeding 8 days decreased root number. In contrast, PG consistently synergised IAA-induced rooting in the dark for contact periods up to 13 days with the highest rooting being recorded at 9 days. An irradiance of 20 W m⁻² from fluorescent lamps halved IAA-induced rooting irrespective of the presence or absence of PG. The culture of shoots at temperatures of 22,25 and 29°C during the root initiation phase (auxin present) and the root emergence phase (auxin absent) produced no difference in rooting response. In the presence of PG the use of liquid culture in place of agar-solidified culture during the auxin-sensitive phase reduced root number but not rooting percentage.     

Ascorbic acid as negative effector of the peroxidase-catalyzed degradation of indole-3-acetic acid

Ascorbic acid is a strong inhibitor of indole-3-acetic oxidation catalyzed by commercial horse-radish peroxidase. In the presence of excess ascorbic acid, the indole-acetic acid oxidation catalysis is apparently blocked. The activity of peroxidase for indoleacetic acid at pH 3.7 and 33°C, in the presence of 2,4-dichlorophenol and MnCl₂ as promotors was measured by polarographic technique. The K_m was 0.27 m M and the maximum velocity was 1.02 mmol O₂ (mg protein)⁻¹ min⁻¹. Dixon plots lead to an apparent K_i of 1.25 (μ M for ascorbic acid and the inhibition was apparently competitive. Ascorbic acid, besides appearing to be a strong inhibitor of the IAA oxidase activity of peroxidase, seemed to protect IAA from total degradation. Addition of more than 5 μ M ascorbic acid produced both an exponential increase in the lag time before the onset of reaction and, at the end, an oxidation protection of 26 μ M IAA when 111 μ M IAA was present at the stawrt. The possibility of ascorbic acid-IAA auxin from endogenous oxidation in plants, is proposed.     

Uptake of Cd2+ and Fe2+ by excised roots of Tamarix aphylla

The uptake of Cd²⁺ by excised roots of Tamarix aphylla (L.) Karst, was investigated using roots of hydroponically grown plants. The concentration isotherm of Cd²⁺ uptake approached saturation with a single phase hyperbola. The time course of Cd²⁺ absorption was generally hyperbolic, with an apparent linear section between 2 and 30 min. The temperature response varied among different temperature ranges: a Q₁₀ of approximately 1.9 was found between 10 and 20°C, but at higher and lower temperatures Q₁₀ values were only 1–1.3. It is concluded that Cd²⁺ uptake by the roots of T. aphylla at moderate temperatures is mediated by a metabolic process, combined with a passive influx component that becomes dominant at higher and lower temperatures. The distribution of the absorption sites for Cd²⁺ and for Fe²⁺ along the roots of T. aphylla was also investigated. Cadmium uptake showed no apparent pattern, whereas a distinct pattern of uptake was observed for Fe²⁺, with the highest rates at the root tip. Iron absorption was stimulated in the presence of nutrients, whereas that of Cd²⁺ was inhibited. Adsorption and absorption of Cd²⁺ were strongly inhibited by Ca²⁺ and by Mg²⁺, but were unaffected by Fe²⁺. Monovalent ions (Na⁺, K⁺, Li⁺) also reduced Cd²⁺ absorption, but to a lesser extent than Ca²⁺ and Mg²⁺. Uptake of Cd⁺ was reduced at lower pH of the medium. The importance of interfering cations for Cd²⁺ tolerance of T. aphylla is emphasized.     

Comparison of the effects of auxin and fusicoccin on phosphate absorption by aged potato tuber discs

Auxin (IAA, 5 × 10⁻⁵ M ) partially prevents the increase in the rate of phosphate uptake during ageing of potato tuber discs ( Solatium tuberosum L. cv. Bintje), whereas fusicoccin (FC, 10⁻⁵ M) stimulates it. After the development of enhanced phosphate transport capacity, the response to fusicoccin is greater than with fresh discs. Complementary experiments on K⁺ (⁸⁶Rb) absorption show that FC also slightly enhances the rate of K⁺ uptake, while IAA has no much effect. It is suggested that IAA acts specifically on the development of a mechanism which occurs during the ageing period, while FC action may be more directly linked to the system of phosphate transport itself.     

Maintenance of the diurnal petiolar movement by exogenous indole-3-acetic acid under day-night cycles in Mimosa pudica

Effects of plant hormones on the diurnal movement of the petiole of Mimosa pudica L. were examined under the conditions of day-night cycles. Surgical removal of the leaf at the middle of the petiole led to gradual loss of the movement. Aqueous solutions of indole-3-acetic acid (IAA) applied as a pulse each day 10 μl at (10⁻⁶−10⁻⁵ M ) or continuously (10⁻⁸−10⁻⁶ M ) to the cut end of the petiole maintained a diurnal movement that has the same phase of oscillation as that shown in the intact leaf. Pulse treatments given at different times of the day had no effect on the phase of the oscillation. Higher concentrations of the acid gave larger amplitudes. Other hormones such as gibberellic acid, kinetin and [R,S]-abscisic acid or 1-aminocyclopropane-1-carboxylic acid did not show diurnal movement-maintaining activities of IAA. Gibberellic acid disturbed the phase of the diurnal movements and kept the petiole elevated at very high positions. A possible action mechanism of IAA is discussed.     

Calcium ion dependency of ethylene production in segments of primary roots of Zea mays

We investigated the effect of Ca²⁺ on ethylene production in 2-cm long apical segments from primary roots of corn ( Zea mays L., B73 × Missouri 17) seedlings. The seedlings were raised under different conditions of Ca²⁺ availability. Low-Ca and high-Ca seedlings were raised by soaking the grains and watering the seedlings with distilled water or 10 m M CaCl₂, respectively. Segments from high-Ca roots produced more than twice as much ethylene as segments from low-Ca roots. Indoleacetic acid (IAA; 1 μ M ) enhanced ethylene production in segments from both low-Ca and high-Ca roots but auxin-induced promotion of ethylene production was consistently higher in segments from high-Ca roots. Addition of I-aminocyclopropane-I-carboxylic acid (ACC) to root segments from low-Ca seedlings doubled total ethylene production and the rate of production remained fairly constant during a 24 h period of monitoring. In segments from high-Ca seedlings ACC also increased total ethylene production but most of the ethylene was produced within the first 6 h. The data suggest that Ca²⁺ enhances the conversion of ACC to ethylene. The terminal 2 mm of the root tip were found to be especially important to ethylene biosynthesis by apical segments and, experiments using ⁴⁵Ca²⁺ as tracer indicated that the apical 2 mm of the root is the region of strongest Ca²⁺ accumulation. Other cations such as Mn²⁺, Mg²⁺, and K⁺ could largely substitute for Ca²⁺. The significance of these findings is discussed with respect to recent evidence for gravity-induced Ca²⁺ redistribution and its relationship to the establishment of asymmetric growth during gravitropic curvature.     

Cyclic AMP-Elevating Agents Inhibit Proliferation of Keratinizing Guinea Pig Epidermal Cells

Cultured guinea pig epidermal cells and dermal fibroblasts were chosen as model systems to study possible growth inhibition by cyclic AMP (cAMP)-elevating drugs. The rate of DNA synthesis was used to assay growth rate in control cultures and those treated with agents which increase intracellular cAMP, including dibutyryl cAMP, the phosphodiesterase inhibitors papaverine and theophylline and agents which stimulate adenylate cyclase, iso-proterenol and prostaglandin E₂ methyl ester. Treatment for 24 h with dibutyryl cAMP (10⁻⁴ to 10⁻² M) inhibited cell growth by 50 to 95%, whereas butyrate(10⁻⁴M) showed essentially no effect. This inhibition could not be attributed to decreased precursor transport or to drug toxicity. Papaverine (10⁻⁶ to 10⁻⁴ M) and theophylline (10⁻⁴ to 10⁻³ M) also gave dose-dependent growth inhibition as did isoproterenol and prostaglandinE₂methyl ester. Radioautographic analysis of grain density after dibutyryl cAMP treatment and ³H-thymidine incorporation indicated no S-phase inhibition. Cyclic AMP-elevating drugs appear to inhibit growth of guinea-pig epidermal cells and dermal flbroblasts by blocking the cell cycle in G₋₂, M₁, or G. ₋₁