Effect of temperature on the dose–response curves for auxin-induced elongation growth in maize coleoptile segments

The dose–response curves for IAA-induced growth in maize coleoptile segments were studied as a function of time and temperature. In addition, the kinetics of growth rate responses at some auxin concentrations and temperatures was also compared. It was found that the dose–response curves for IAA-induced elongation growth were, independently of time and temperature, bell-shaped with an optimal concentration at 10⁻⁵ M IAA. The kinetics of IAA-induced growth rate responses depended on IAA concentration and temperature, and could be separated into two phases (biphasic reaction). The first phase (very rapid) was followed by a long lasting one (second phase), which began about 30 min after auxin addition. For coleoptile segments incubated at 30°C, the amplitudes of the first and second phase were significantly higher, when compared with 25°C, at all IAA concentrations studied. However, when coleoptile segments were incubated at 20°C, the elongation growth of coleoptile segments treated with suboptimal IAA concentrations was diminished, mainly as a result of both phases reduction. In conclusion, we propose that the shape of the dose–response curves for IAA-induced growth in maize coleoptile segments is connected with biphasic kinetic of growth rate response.     

Importance of Time after Excision and of pH on the Kinetics of Response of Wheat Coleoptile Segments to Added Indoleacetic Acid

Segments of coleoptiles of 3-day-old wheat (Triticum x Aestivum L. cv. Kharkov M.C. 22) grown at 24 C were strung on a glass rod and the kinetics of their elongation in 0.01 m K-phosphate buffer was examined photometrically. Measured rates of elongation in response to treatments were corrected by subtraction of endogenous rates. The customary practice of testing the effects of growth regulators added between the two endogenous surges of growth, that is, up to 3 hours after segments were excised from coleoptiles, gave erroneous kinetic data. Rates of response were then limited by the passive penetration of added auxin and the second endogenous surge interfered with late responses. It was necessary to wait for a phase of more rapid but more steady elongation after the second endogenous surge was over, about 4 hours for wheat at 25 C, to attain the active uptake required for nearly synchronous response through the segment. The more active uptake in this steady phase was confirmed with beta-[2-(14)C]indoleacetic acid and it was greater at pH 5 than at pH 7. The degree of dissociation of indoleacetic acid added at pH 7 was an impediment to penetration that could be compensated for by removal of intercellular air. The pH did not influence the endogenous rate of elongation. The dependence of the rate of elongation on the concentration of indoleacetic acid added at pH 5 was bell-shaped with maximum rate at 10 mum indoleacetic acid, in confirmation of previous measurements made over long intervals of time. The relation between the response and suboptimal concentrations was not sigmoid but was indicative of greater binding affinity than previously reported.     

Fusicoccin-induced growth and hydrogen ion excretion of Avena coleoptiles: Relation to auxin responses

Summary The fungal toxin fusicoccin (FC) induces both rapid cell elongation and H⁺-excretion in Avena coleoptiles. The rates for both responses are greater with FC than with optimal auxin, and in both cases the lag after addition of the hormone is less with FC. This provides additional support for the acid-growth theory. The FC responses resemble the auxin responses in that they are inhibited by a range of metabolic inhibitors, but the responses differ in three ways. First auxin, but not FC, requires continual protein synthesis for its action. The auxin-induced H⁺-excretion is inhibited by water stress or by low external pH, while the FC-induced H⁺-excretion is much less sensitive to either. It is concluded that auxin-induced and FC-induced H⁺-excretion may occur via different mechanisms.Abbreviations FC fusicoccin - DNP dinitrophenol - CCCP carbonylcyanide m-chlorophenylhydrazone - CHl cycloheximide - IAA indoleacetic acid     

Evidence that Auxin-induced Growth of Soybean Hypocotyls Involves Proton Excretion

The role of H⁺ excretion in auxin-induced growth of soybean hypocotyl tissues has been investigated, using tissues whose cuticle was rendered permeable to protons or buffers by scarification (scrubbing). Indoleacetic acid induces both elongation and H⁺ excretion after a lag of 10 to 12 minutes. Cycloheximide inhibits growth and causes the tissues to remove protons from the medium. Neutral buffers (pH 7.0) inhibit auxin-induced growth of scrubbed but not intact sections; the inhibition increases as the buffer strength is increased. Both live and frozen-thawed sections, in the absence of auxin, extend in response to exogenously supplied protons. Fusicoccin induces both elongation and H⁺ excretion at rates greater than does auxin. These results indicate that H⁺ excretion is involved in the initiation of auxin-induced elongation in soybean hypocotyl tissue.     

Comparison of Auxin-induced and Acid-induced Elongation in Soybean Hypocotyl

Acid-induced growth was compared to auxin-induced growth. After a transient pH 4-induced increase in the elongation rate was completed, auxin could still induce an enhanced rate of elongation in soybean (Glycine max) hypocotyl segments. This auxin response occurred both when the medium was changed to pH 6 before auxin addition, and when the auxin was added directly to the pH 4 medium. This postacid response to auxin was persistent, and quite unlike a postacid response to acid, which was again shortlived. One mm N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7) inhibited the first response to auxin (the first response to auxin being similar to the acid response), but not the second response. This did not appear to be simply a hydrogen ion neutralizing effect, however, since a 50-fold increase in buffer concentration at pH 6 did not inhibit the first response. Decrease in the pH of the external medium, previously shown to occur with excised soybean hypocotyl segments, was not affected by auxin. Furthermore, this pH drop, during which the cells appear to be adjusting their external pH to about 5.4, did not result in an increased rate of elongation. Addition of auxin after the equilibrium pH had been attained did not alter the pH, but it did increase the rate of elongation, eliciting a normal auxin response. It was concluded that hydrogen ions do not mediate in long term auxin-induced elongation in soybean hypocotyl.     

Genetic Dissection of the Relative Roles of Auxin and Gibberellin in the Regulation of Stem Elongation in Intact Light-Grown Peas

Exogenous gibberellin (GA) and auxin (indoleacetic acid [IAA]) strongly stimulated stem elongation in dwarf GA1-deficient le mutants of light-grown pea (Pisum sativum L.): IAA elicited a sharp increase in growth rate after 20 min followed by a slow decline; the GA response had a longer lag (3 h) and growth increased gradually with time. These responses were additive. The effect of GA was mainly in internodes less than 25% expanded, whereas that of IAA was in the older, elongating internodes. IAA stimulated growth by cell extension; GA stimulated growth by an increase in cell length and cell number. Dwarf lkb GA-response-mutant plants elongated poorly in response to GA (accounted for by an increase in cell number) but were very responsive to IAA. GA produced a substantial elongation in lkb plants only in the presence of IAA. Because lkb plants contain low levels of IAA, growth suppression in dwarf lkb mutants seems to be due to a deficiency in endogenous auxin. GA may enhance the auxin induction of cell elongation but cannot promote elongation in the absence of auxin. The effect of GA may, in part, be mediated by auxin. Auxin and GA control separate processes that together contribute to stem elongation. A deficiency in either leads to a dwarfed phenotype.     

Growth Inhibition and Recovery in Roots Following Temporary Treatment with Auxin

Continuous recording (streak photography) of elongation of roots treated with IAA (10^–6–10^–7M) showed that removal of IAA from the nutrient solution resulted in a rapid resumption of elongation, unless the IAA treatment was shorter than 60 min. If it was shorter, the recovery was delayed, so that it occurred about 1 hour after the beginning of the treatment, independently of the duration of the treatment, down to 4 min. This behavior of roots was observed in all the species investigated (corn, pea, sunflower, onion), also in response to NAA and 2,4-D. This time lag in recovery of elongation after brief auxin treatment is discussed in connection with the radial concentration gradient of auxin in the root imposed by external auxin. The possible role of a radial gradient of auxin (concentration decreasing with distance from the center) in the control of root elongation is suggested.     

Oscillation in the Growth Rate and a Shortening of the Lag Time for IAA Response by Cotyledons, Gibberellic Acid and Sucrose in Bean Hypocotyls

Oscillation in the growth rate as well the lag time for an IAAresponse in bean (Phaseolus vulgaris) hypocotyls were studiedwith a displacement transducer. The growing zone of the hypocotylshowed continuous oscillation in the growth rates for intactseedlings, hypocotyl explants with or without cotyledons, andexplants submerged in water. The lag time for the IAA response was shortened by the presenceof cotyledon tissues as well as by preliminary treatment withgibberellic acid and sucrose. When IAA first was applied tothe basal cut surface then to the upper cut surface of the hypocotylexplant, two growth responses with distinct lag periods werefound. The difference in the passage of auxin due to the directionof transport in the hypocotyl is discussed. (Received February 9, 1983; Accepted August 9, 1983)     

Auxin Transport Is Required for Hypocotyl Elongation in Light-Grown but Not Dark-Grown Arabidopsis

Many auxin responses are dependent on redistribution and/or polar transport of indoleacetic acid. Polar transport of auxin can be inhibited through the application of phytotropins such as 1-naphthylphthalamic acid (NPA). When Arabidopsis thaliana seedlings were grown in the light on medium containing 1.0 μm NPA, hypocotyl and root elongation and gravitropism were strongly inhibited. When grown in darkness, however, NPA disrupted the gravity response but did not affect elongation. The extent of inhibition of hypocotyl elongation by NPA increased in a fluence-rate-dependent manner to a maximum of about 75% inhibition at 50 μmol m⁻² s⁻¹ of white light. Plants grown under continuous blue or far-red light showed NPA-induced hypocotyl inhibition similar to that of white-light-grown plants. Plants grown under continuous red light showed less NPA-induced inhibition. Analysis of photoreceptor mutants indicates the involvement of phytochrome and cryptochrome in mediating this NPA response. Hypocotyls of some auxin-resistant mutants had decreased sensitivity to NPA in the light, but etiolated seedlings of these mutants were similar in length to the wild type. These results indicate that light has a significant effect on NPA-induced inhibition in Arabidopsis, and suggest that auxin has a more important role in elongation responses in light-grown than in dark-grown seedlings.     

Early Responses of Excised Stem Segments to Auxins

The elongation rate of lupin hypocotyl and pea stem segmentswas measured every minute after the addition of various auxinsand auxin precursors. After a latent period the growth-rateincreased to a peak, fell to a minimum, and with most compoundsincreased to a second maximum. Compounds used include indol-3yl-aceticacid (IAA), indol-3yl-acetamide, naphth-2yl-oxyacetic acid,naphth- lyl-acetamide, and indol-3yl butyric acid. The extensibilityof the cell walls of the lupin segments was measured with anInstron Universal Testing Instrument at intervals after theaddition of IAA and it was shown that the lag period beforethe extensibility increased was longer than that for the growth-rate. Kinetic studies were made of the effect of Actinomycin D onIAA-induced growth. RNA synthesis during the first 20 and 40min after IAA addition was also examined in segments exposedto labelled RNA precursors during these times. The results supportthe conclusion that RNA synthesis is not necessary for the initialaction of auxin on elongation.     

INTERACTION OF SUGARS AND AUXINS IN PEA EPICOTYL SECTION GROWTH

Purves , W. K., and A. W. Galston . (Yale U., New Haven, Conn.) Interaction of sugars and auxins in pea epicotyl section growth. Amer. Jour. Bot. 47 (8): 665–669. Illus. 1963.—The nature and magnitude of the response of “S₁” etiolated pea-epicotyl sections to auxin are determined by the concentration of sugar in the growth medium. For example, the concentration of IAA inducing maximum elongation shifts through at least 3 orders of magnitude in response to varying sucrose concentrations, from ca. 10^–4 M, with no sucrose, to 10^–7 M, with 2% sucrose. Similarly, the inhibitory action of high levels of IAA on elongation occurs only in the presence of sucrose. By contrast, although sucrose also promotes water uptake, it affects the IAA optimum for this process only slightly. The action of IAA on growth can be detected immediately, but the growth response to sucrose occurs only after a 6–8 hr. lag. If tissues are supplied with sucrose, then 1-hr. exposures to IAA can be as effective on growth as continuous 20-hr. exposures, depending on the time at which such exposures are given. Thus, 10^—4 M IAA applied in the presence of 2% sucrose is markedly inhibitory to elongation in hours 1–3, relatively inactive in hours 4–6, and strongly promotive after hour 7. The change from inhibitory to promotive action thus coincides in time with the length of the lag period for sucrose action.     

Growth rates and auxin effects in graviresponding gynophores of the peanut, Arachis hypogaea (Fabaceae)

The gynophore of the peanut plant (Arachis hypogaea) is a specialized organ that carries and buries the fertilized ovules into the soil in order for seed and fruit development to occur underground. The rates of growth of vertically and horizontally oriented gynophores were measured using a time-lapse video imaging system. We found that the region of maximum extension growth due to elongation (termed the Central Elongation Zone) is located on average at 2-5 mm from the tip. In the first 0-4 h after horizontal reorientation (gravistimulation), new zones of growth emerge on the upper surface, while the elongation zone of the lower side decreases in size and magnitude. Four to six hours after reorientation the zones of maximum growth are almost equal in size and location on the upper and lower sides. The growth rate and the gravitropic response decreased dramatically, upon the excision of the ovule region (terminal 1.5 mm), but a gravitropic growth response could be restored by applying the auxin indole-3-acetic acid exogenously to the excised tip. The addition of napthylphthalamic acid (an auxin transport inhibitor) at the ovule region allowed some growth to occur, but the gynophores do not respond normally to gravity, upon horizontal reorientation. We discuss the role of auxin in the gravitropic response of the gynophore.     

Rapid-growth responses of corn root segments: Effect of auxin on elongation

The effect of indole-3-acetic acid (IAA) on the elongation rates of 2 mm corn (Zea mays L.) root segments induced by citrate-phosphate buffer (or unbuffered) solutions of pH 4.0 and 7.0 was studied. At pH 7.0, auxin initially reduced the elongation rate in both buffered and unbuffered solutions. Only in buffer at pH 7.0 was auxin at a concentration of 0.1 M found to promote the elongation rate though briefly. THis promoted rate represented only ca. 20% of the rate achieved with only buffer at pH 4.0. Auxin in pH 4.0 buffered and unbuffered solutions only served to reduce the elongation rates of root segments. Some comparative experiments were done using 2 mm corn coleoptile segments. Auxin (pH 6.8) promoted the elongation rate of coleoptile segments to a level equal or greater than the maximal H ion-induced rate. The two responses of root segments to auxin are compared to auxin action in coleoptile growth.     

Critical consideration on the relationship between auxin transport and calcium transients in gravity perception of Arabidopsis seedlings

Plants regulate their growth and morphogenesis in response to gravity field, known as gravitropism. In the early process of gravitropism, changes in the gravity vector (gravistimulation) are transduced into certain intracellular signals, termed gravity perception. The plant hormone auxin is not only a crucial factor to represent gravitropism but also a potential signaling molecule for gravity perception. Another strong candidate for the signaling molecule is calcium ion of which cytoplasmic concentration ([Ca²⁺]_c) is known to increase in response to gravistimulation. However, relationship between these two factors, say which is in the first place, has been controversial. This issue is addressed here mainly based on recent progress including our latest studies. Gravistimulation by turning plants 180° induced a two-peaked [Ca²⁺]_c-increase lasting for several minutes in Arabidopsis seedlings expressing apoaequorin; only the second peak was sensitive to the gravistimulation. Peak amplitudes of the [Ca²⁺]_c-increase were attenuated by the 10 µM auxin transport inhibitor (TIBA) and vesicle trafficking inhibitor (BFA), whereas the onset time and rate of rise of the second peak were not significantly altered. This result indicates that polar auxin transport is not involved in the initial phase of the second [Ca²⁺]_c-increase. It is likely that the gravi-induced [Ca²⁺]_c-increase constitutes an upstream event of the auxin transport, but may positively be modulated by auxin since its peak amplitude is attenuated by the inhibition of auxin transport.Key words: auxin, calcium, gravity perception, gravitropism, pin-formed (PIN) protein, Arabidopsis thaliana     

Induction of elongation in maize coleoptiles by hexachloroiridate and its interrelation with auxin and fusicoccin action

The demonstration of an auxin-stimulated NADH-oxidase in the plasma membrane (Brightman et al. 1988. Plant Physiol. 86: 1264–1269) has led to the suggestion that the plasma membrane redox system is involved in the mechanism of auxin action. To evaluate the relevance of this concept in vivo, the influence of micromolar concentrations of hexachloroiridate (IV), an impermeable electron acceptor for the plant plasma membrane redox system, on elongation growth of excised, abraded maize coleoptile ( Zea mays L. cv. Golden Bantam) segments was studied. It was found that the substance induced a rapid growth response if the experiment was carried out in an unbuffered solution. This effect was entirely prevented by a 2 m M phosphate buffer. Nevertheless, the acid-growth-theory does not seem sufficient to explain this effect, since proton extrusion is induced without a lag, whereas increased growth rates commence after a lag phase of 40 min.
If growth is stimulated by a pretreatment with fusicoccin or auxin, hexachloroiridate IV transiently inhibits growth. The kinetics of the response are then determined by the concentrations of hexachloroiridate and auxin or fusicoccin. These results are compatible with the view that the plasma membrane redox system is somehow involved in the control of elongation growth.     

Responses of Avena coleoptiles to suboptimal fusicoccin: kinetics and comparisons with indoleacetic Acid

Proton excretion induced by optimal concentrations of indoleacetic acid (IAA) and fusicoccin (FC) differs not only in maximum rate of acidification but also in the lag before onset of H⁺ excretion and in sensitivity to cycloheximide. Because these differences might simply be a consequence of the difference in rate of proton excretion, FC and IAA have now been compared using oat coleoptiles (cv. Victory) under conditions where the rates of acidification are more similar, i.e. suboptimal FC versus optimal IAA. As the concentration of FC is reduced, the rate of H⁺ excretion decreases, the final equilibrium pH increases, and the lag before detectable acidification increases up to 7-fold. This enhanced lag period is not primarily a consequence of wall buffering, inasmuch as it persists when a low concentration of FC is added to sections which were already excreting H⁺ in response to IAA. An extended lag also occurs, upon reduction of FC levels, in the hyperpolarization of the membrane potential, before enhancement of O₂ uptake and before the increased rate of Rb⁺ uptake. The presence or absence of a lag is not a distinguishing feature between FC and IAA actions on H⁺ excretion and cannot be used to discriminate between their sites of action. In contrast, the insensitivity of FC-induced H⁺ excretion to cycloheximide, as compared with the nearly complete inhibition of this auxin effect by cycloheximide, persists even at dilute concentrations of FC. This seems to be a basic difference in H⁺ excretion by IAA and FC.     

IAA-Induced Growth Responses of Decapitated Corn Seedlings: Indications of Two Apparent Adaptations with a Possible Role in Gravitropism

The vertical growth responses of corn seedlings (Zea mays L. Mo17 × B73) were determined over an 8-hour period. When seedlings were decapitated 3 millimeters from the coleoptile's tip and supplied with indole-3-acetic acid (IAA) in 1.5% agar blocks, the response was dependent both on time and IAA concentration. The dose-response curves changed in shape and magnitude depending on the total time of IAA application. High concentrations (>3.2 × 10⁻⁶ molar) initially produced high relative growth rates that decreased back to the intact rate (0.03 millimeter per hour per millimeter) after 3 hours. Low concentrations (<1.0 × 10⁻⁶ molar), or agar blocks without IAA, resulted in a rapid decrease from the intact rate to a level that stabilized at 0.01 millimeter per hour per millimeter until the growth rate began to recover after 3 to 4 hours. Intermediate concentrations produced responses similar to that of the intact organ, though some features of these responses were unique.

The coleoptile curvature in response to gravity depended upon whether the coleoptiles were intact, decapitated, or decapitated and supplied with IAA. Coleoptiles decapitated and not supplied wth IAA showed little or no curvature for 3 hours after decapitation. By this time an adaptation, evoked by the low IAA level, had developed and the coleoptiles began to curve steadily. When 1.0 or 3.2 × 10⁻⁶ molar IAA was supplied, curvature was initiated within the first 30 minutes and reached a maximum rate before decreasing and stopping after 3 to 4 hours. The sequence of events in response to these concentrations was similar to the intact sequence but the curvature rate was reduced to one-third to one-half. A model for the autotropic response involving an auxin concentration-dependent, growth-modulating mechanism capable of two modes of adaptation is described.

     

Effects of the steroidal alkaloid tomatine in auxin bioassays and its interaction with indole-3-acetic acid

Summary The steroidal alkaloid tomatine did not enhance elongation of oat coleoptile and first internode sections, or of wheat coleoptile sections. Higher concentrations of the alkaloid inhibited elongation and interacted antagonistically with IAA. Although 10^-4 M tomatine alone did not influence elongation of oat coleoptile sections, it did reduce growth response to exogenous IAA. Tomatine concentrations less than 10^-4 M did not influence response to IAA. The auxin activity of tomatine, reported by Vendrig, was therefore not confirmed.     

The Inhibition by Cytokinin of Auxin-Promoted Elongation in Excised Soybean Hypocotyl

The experiments characterize the inhibition by kinetin of auxin-promoted elongation in excised hypocotyl sections of 3-day soybean seedlings (Glycine max cv. Hawkeye 63). It was found that concentrations of kinetin above 4.2 μM did not further inhibit auxin-promoted elongation. Kinetin is as potent an inhibitor of elongation as actinomycin D or cycloheximide. Tissue incubated for 3 or 5 h in the absence of auxin or cytokinin would, upon addition of auxin, exhibit a new growth rate similar to that of tissue grown in auxin for the entire incubation period. Similarly, tissue grown for 3 and 5 h in the presence of auxin would revert to the control rate of elongation upon addition of kinetin. A 10 to 30 min preincubation in kinetin yielded the tissue incapable, for the ensuing 6 h, of increasing its rate of elongation in response to auxin. Zeatin and isopentenyladenine were more potent than kinetin and benzyladenine in the inhibition of elongation. Levels of ethylene produced in the presence of auxin plus cytokinin indicated that it was not involved in this auxin-cytokinin interaction. Kinetin by itself did not promote elongation; nor did it enhance auxin-promoted elongation at low auxin concentrations.     

Blue-light-mediated shade avoidance requires combined auxin and brassinosteroid action in Arabidopsis seedlings

Plant growth in dense vegetation can be strongly affected by competition for light between neighbours. These neighbours can not only be detected through phytochrome-mediated perception of a reduced red:far-red ratio, but also through altered blue light fluence rates. A reduction in blue light (low blue) induces a set of phenotypic traits, such as shoot elongation, to consolidate light capture; these are called shade avoidance responses. Here we show that both auxin and brassinosteroids (BR) play an important role in the regulation of enhanced hypocotyl elongation of Arabidopsis seedlings in response to blue light depletion. Only when both hormones are experimentally blocked simultaneously, using mutants and chemical inhibitors, will the response be fully inhibited. Upon exposure to low blue several members of the cell wall modifying XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE (XTH) protein family are regulated as well. Interestingly, auxin and BR each regulate a subset of these XTHs, by which they could regulate cell elongation. We hypothesize that auxin and BR regulate specific XTH genes in a non-redundant and non-synergistic manner during low-blue-induced shade avoidance responses of Arabidopsis seedlings, which explains why both hormones are required for an intact low-blue response.