Galactose prevents proton excretion but not IAA-induced growth in segments of azuki bean epicotyls

We investigated the effect of galactose on IAA-induced elongation and proton excretion in azuki bean (Vigna angularis Ohwi et Ohashi) segments in order to confirm whether or not protons were involved in auxin-induced growth. Galactose inhibited the IAA-induced decrease in the solution pH but had no inhibitory effect on IAA-induced growth in segments of azuki bean epicotyls. On the other hand, galactose inhibited both IAA-induced growth and proton excretion in oat (Avena sativa L.) coleoptile segments. From these results it is unlikely that IAA-induced growth is mediated by proton excretion at least in azuki bean epicotyls.Abbreviations IAA indole-3-acetic acid - FC fusicoccin     

Evidence for the acid-growth theory of fusicoccin action

Three predictions of the acid-growth theory of fusicoccin (FC) action in inducing cell elongation were reinvestigated using abraded segments of maize (Zea mays L.) coleoptiles. i) Quantitative comparison of segment elongation and medium-acidification kinetics measured in the same sample of tissue shows that these FC-induced processes are strictly correlated in time and respond coordinately to cations present in the medium. ii) Fusicoccin (1 mol l^-1) induces a rapid acidification of the cell-wall solution, reaching a final level of pH 3.8–4.0. Exogenous protons are able to substitute quantitatively for FC in causing segment elongation at pH 3.8–4.0. At pH 4, FC has no additional effect on cell elongation. iii) Neutral buffers (pH 7) completely abolish the FC-mediated growth response. iv) Cycloheximide (10 mg l^-1) inhibits both FC-induced and acid-buffer(pH 4)-induced elongation after a lag of 40–45 min, and FC-induced H⁺ excretion after a lag of 2 h. Under the same conditions, indole-3-acetic acid-induced elongation and H⁺ excretion are inhibited without detectable lag. It is concluded that these results are fully compatible with the acid-growth theory of FC action.Abbreviations IAA indole-3-acetic acid - CHI cycloheximide - FC fusicoccin     

Evidence against the acid-growth theory of auxin action

Four experimental predictions of the acid-growth theory of auxin (indole-3-acetic acid, IAA) action in inducing cell elongation were reinvestigated using abraded segments of maize (Zea mays L.) coleoptiles. i) Quantitative comparison of segment elongation and medium-acidification kinetics measured in the same sample of tissue reveals that these IAA-induced processes are neither correlated in time nor responding coordinately to cations present in the medium. ii) Exogenous protons are not able to substitute for IAA in causing segment elongation at the predicted pH of 4.5–5.0. Instead, external buffers induce significant segment elongation only below pH 4.5, reaching a maximal response at pH 1.75–2.5. Acid and IAA coact additively, and therefore independently, in the whole range of feasible pH values. iii) Neutral or alkaline buffers (pH 6–10) are unable to abolish the IAA-mediated growth response and have no effect on its lag-phase. iv) Fusicoccin, at a concentration producing the same H⁺ excretion as high concentrations of IAA, is ineffective in inducing segment elongation. Moreover, sucrose and other sugars can quantiatively substritute for IAA in inducing H⁺ excretion but are likewise ineffective in inducing elongation. It is concluded that these results are incompatible with the acid-growth theory of auxin action.Abbreviations IAA indole-3-acetic acid - FC fusicoccin     

Effect of cycloheximide on IAA-induced proton excretion and IAA-induced growth in abraded Avena coleoptiles

IAA-induced proton excretion in peeled or abraded oat ( Avena saliva L. cv. Victory) coleoptiles is closely associated with IAA-induced growth. It was attempted to separate these two processes by using cycloheximide to inhibit them differentially. Growth of abraded coleoptile segments was measured by a shadow graphic method, and their IAA-induced acidification of the external solution was monitored with a pH meter. IAA stimulated proton excretion in abraded Avena coleoptile segments after a 13 min lag. IAA-induced proton excretion was inhibited within 5 min by cycloheximide at concentrations of 1.8 × 10⁻⁶, 3.6 × 10 or 3.6 × 10⁻⁵ M. Cycloheximide at these concentrations, added within 4 min of IAA, prevented IAA-induced acidification of the medium for at least 60 min. However, it did not prevent IAA-induced growth during this time. It is concluded that some of the initial IAA-induced growth seen in Avena coleoptiles is independent of detectable IAA-induced proton excretion.     

Effect of diclofop-methyl and 2,4-D on transmembrane proton gradient: a mechanism for their antagonistic interaction

The site of action of the postemergence graminicide, diclofop-methyl (DM), in susceptible plants is possibly the plasmalemma. Indole-acetic acid (IAA)- and fusicoccin (FC)-induced net proton excretion in Avena coleoptiles was inhibited by the free acid, diclofop. However, net proton excretion recovered within 2 h when 2,4-dichlorophenoxy acid (2,4-D) was added simultaneously with diclofop. Diclofop depolarized the membrane potential (Em) within 12 min but the Em recovered within 30 min when diclofop was removed and replaced with either IAA or 2,4-D. The inhibition of IAA-induced coleoptile growth by DM and the membrane effects of its acid, diclofop, were partially reversed by 2,4-D if it was added shortly after treatment of the tissue. These results are consistent with the reversal of DM injury in whole plants with 2,4-D.     

Flower stalk segments of Arabidopsis thaliana ecotype Columbia lack the capacity to grow in response to exogenously applied auxin

Exogenously applied IAA stimulated cell elongation of segments excised from flower stalks of Arabidopsis thaliana ecotype Landsberg erecta (Ler) by increasing the cell wall extensibility, but it did not affect that of ecotype Columbia (Col). Treatment with a low pH buffer solution (pH 4.0) or fusicoccin (FC), a reagent activating H(+)-ATPases, significantly increased the cell wall extensibility and promoted elongation growth of flower stalk segments of both ecotypes, indicating that the flower stalk segments of Col possess the capacity to grow under acidic pH conditions. IAA promoted the proton excretion in segments of Ler but not of Col. On the other hand, FC increased the proton excretion in segments of Col as much as that of Ler. These results suggest that IAA activates the plasma membrane H(+)-ATPases in the segments of Ler but not those of Col, while FC activates them in both ecotypes. Flower stalks of Col may lack the mechanisms of activation by IAA of the plasma membrane H(+)-ATPases.     

Reexamination of the Acid growth theory of auxin action

Some crucial arguments against the acid growth theory of auxin action (U Kutschera, P Schopfer [1985] Planta 163: 483-493) have been reinvestigated by simultaneous measurements of proton fluxes and growth of maize (Zea mays L.) coleoptiles. Special care was taken to obtain a mild, effective, and reproducible abrasion of the cuticle. Proton secretion rates were determined in a computer-controlled pH-stat. In some experiments, equilibrium pH was measured. Growth rates were determined simultaneously in the same vessel using a transducer-type auxanometer. It was found that (a) the timing of auxin and fusicoccin-induced (FC) proton secretion and growth matches well, (b) the equilibrum external pHs in the presence of IAA and FC are lower than previously recorded and below the so-called `threshold-pH,' (c) neutral or alkaline unbuffered solutions partially inhibit FC and IAA-induced growth in a similar manner, (d) the action of pH, FC, and IAA on growth are not additive. It is concluded that the acid-growth-theory correctly describes incidents taking place in the early phases of auxin-induced growth.     

Role of chloride ions in the promotion of auxin-induced growth of maize coleoptile segments

Background and Aims

The mechanism of auxin action on ion transport in growing cells has not been determined in detail. In particular, little is known about the role of chloride in the auxin-induced growth of coleoptile cells. Moreover, the data that do exist in the literature are controversial. This study describes experiments that were carried out with maize (Zea mays) coleoptile segments, this being a classical model system for studies of plant cell elongation growth.

Methods

Growth kinetics or growth and pH changes were recorded in maize coleoptiles using two independent measuring systems. The growth rate of the segments was measured simultaneously with medium pH changes. Membrane potential changes in parenchymal cells of the segments were also determined for chosen variants. The question of whether anion transport is involved in auxin-induced growth of maize coleoptile segments was primarily studied using anion channel blockers [anthracene-9-carboxylic acid (A-9-C) and 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS)]. In addition, experiments in which KCl was replaced by KNO₃ were also performed.

Key Results

Both anion channel blockers, added at 0·1 mm, diminished indole-3-acetic acid (IAA)-induced elongation growth by ∼30 %. Medium pH changes measured simultaneously with growth indicated that while DIDS stopped IAA-induced proton extrusion, A-9-C diminished it by only 50 %. Addition of A-9-C to medium containing 1 mm KCl did not affect the characteristic kinetics of IAA-induced membrane potential changes, while in the presence of 10 mm KCl the channel blocker stopped IAA-induced membrane hyperpolarization. Replacement of KCl with KNO₃ significantly decreased IAA-induced growth and inhibited proton extrusion. In contrast to the KCl concentration, the concentration of KNO₃ did not affect the growth-stimulatory effect of IAA. For comparison, the effects of the cation channel blocker tetraethylammonium chloride (TEA-Cl) on IAA-induced growth and proton extrusion were also determined. TEA-Cl, added 1 h before IAA, caused reduction of growth by 49·9 % and inhibition of proton extrusion.

Conclusions

These results suggest that Cl^– plays a role in the IAA-induced growth of maize coleoptile segments. A possible mechanism for Cl^– uptake during IAA-induced growth is proposed in which uptake of K⁺ and Cl^– ions in concert with IAA-induced plasma membrane H⁺-ATPase activity changes the membrane potential to a value needed for turgor adjustment during the growth of maize coleoptile cells.     

Characteristics and implications of prolonged fusicoccin-induced growth of Avena coleoptile sections

A study has been made of the prolonged growth of Avena coleoptile sections in response to fusicoccin (FC), a phytotoxin that promotes apoplastic acidification. The final amount of FC-induced growth is a function of the FC concentration. Removal of the epidermis speeds up the initial rate of elongation and shortens the duration of the response, without affecting the total amount of extension. A suboptimal FC concentration (7×10⁻⁸ M ) which induces the same rate of proton excretion as does optimal indoleacetic acid (IAA) (1×10⁻⁵ M ), causes elongation which is 60–75% of that induced by IAA in 4 h or 50–65% in 7 h. This suggests that acid-induced extension could make a major contribution to auxin-induced growth for at least 7 h.     

Role of protein and RNA synthesis in the initiation of auxin-mediated growth in coleoptiles of Zea mays L.

The kinetics of inhibition by protein- and RNA-synthesis inhibitors (cycloheximide and cordycepin, respectively) of indole-3-acetic acid (IAA)-induced elongation growth were investigated using abraded coleoptile segments of Zea mays L. Removal of the cuticle — a diffusion barrier for solutes — by mechanical abrasion of the outer epidermal cell wall increased the effectiveness of inhibitors tremendously. In an attempt to elucidate the role of growth-limiting protein(s) (GLP) in the growth mechanism the following results were obtained. The elongation induced by IAA was completely inhibited when cycloheximide (10 mol·l^-1) was applied to abraded coleoptile segments as shortly as 10 min before the onset of the growth response (=5 min after administration of IAA). However, when cycloheximide was applied after 60 min of IAA treatment (when a steady-state growth rate is reached), the time required for complete cessation of growth was much longer (about 40 min). Cycloheximide inhibited the incorporation of [³H]leucine into protein within about 5 min. Cordycepin (400 mol·l^-1) prevented IAA-induced growth when applied as shortly as 25 min before the onset of the growth response (=10 min before administration of IAA) but required more than 60 min for a full inhibition of steady-state growth. The incorporation of [³H]adenosine into RNA was inhibited by cordycepin within 10 min. It is concluded that, contrary to previous investigations with nonabraded organ segments, the initiation of growth by IAA depends directly on the synthesis of GLP. Moreover, the apparent lifetime of GLP is at least four times longer than the time required by cycloheximide to inhibit the initiation of growth by IAA. This is interpreted to mean that GLP is not present before IAA starts to act but is synthesized as a consequence of IAA action starting a few minutes before the initiation of growth. Interpreting the kinetics of growth inhibition by cordycepin in a similar way, we further conclude that GLP synthesis is mediated by IAA-induced synthesis of the corresponding mRNA which starts about 10 min before the onset of GLP synthesis. Inhibition by cycloheximide and cordycepin of IAA-induced growth cannot be alleviated by acidifying the cell wall to pH 4-5, indicating that these inhibitors do not act on growth via an inhibition of auxin-mediated proton excretion.Abbreviations CHI cycloheximide - COR cordycepin - GLP growth-dimiting protein(s) - IAA indole-3-acetic acid - mRNA_GLP mRNA coding for GLP     

Fusicoccin counteracts inhibitory effects of high temperature on auxin-induced growth and proton extrusion in maize coleoptile segments

Plant growth and development are tightly regulated by both plant growth substances and environmental factors such as temperature. Taking into account the above, it was reasonable to point out that indole-3-acetic acid (IAA), the most abundant type of auxin in plants, could be involved in temperature- dependent growth of plant cells. We have recently shown that growth of maize coleoptile segments in the presence of auxin (IAA) and fusicoccin (FC) shows the maximum value in the range 30–35°C and 35–40°C, respectively. Furthermore, simultaneous measurements of growth and external medium pH indicated that FC at stressful temperatures was not only much more active in the stimulation of growth, but was also more effective in acidifying the external medium than IAA. The aim of this addendum is to determine interrelations between the action of IAA and FC (applied together with IAA) on growth and medium pH of maize coleoptile segments incubated at high temperature (40°C), which was optimal for FC but not for IAA.Key words: auxin, fusicoccin, coleoptile segments, elongation growth, medium pHA well studied aspect of auxin action especially in maize coleoptile, is its effect on cell elongation, proton extrusion and membrane potential.^1–7 It is now generally agreed that indole-3-acetic acid (IAA), as the principal regulator of plant elongation growth, causes (i) acceleration of elongation growth as compared to endogenous growth, (ii) enhancement of proton extrusion as compared to auxin—free medium, and (iii) transient depolarization followed by a slow hyperpolarization of membrane potential. According to the “acid growth theory” of elongation growth,^8–11 auxin induced cell wall acidification provides favorable conditions for cell wall loosening, a requirement for cell elongation. At least in maize coleoptile segments, auxin induced cell wall acidification is mediated by increased activity and/or amount of the PM H⁺-ATPase.^11,12 In the case of fusicoccin, which mimics the effect of auxin in many respects,¹³ it was shown that FC-binding site arises from interaction of the 14-3-3 protein dimmer with the C-terminal autoinhibitory domain of the H⁺-ATPase and that FC stabilizes this complex.^14–18 It should be pointed out that in spite of abundant literature on the mechanism through which IAA or FC control growth of grass coleoptiles, little is know how these substances work at extreme temperatures. Over the past decade, the involvement of 14-3-3 proteins in plant stress responses has often been suggested.¹⁹ For example, work by Chelysheva et al.,²⁰ and Babakov et al.,²¹ demonstrated that under low temperature and high osmolarity conditions, 14-3-3 proteins interact with the C-terminal autoinhibitory domain of the PM H⁺-ATPase activating the proton pump that play a key role in stress responses in higher plants. We have recently shown²² that FC at 40°C induced maximal growth whereas growth observed at the same temperature in the presence of IAA was reduced by 33% compared to the maximal value at 30°C. It was also found²² that at 40°C the kinetics of the pH change differed significantly for both growth substances; the segments treated with IAA at 40°C were virtually not able to acidify the external medium, whereas FC at this temperature caused practically maximal acidification. In this addendum we have shown that application of FC together with IAA conteracted the inhibitory effect of high temperature (40°C) on IAA-induced growth and proton extrusion in maize coleoptile segments (Fig. 1). For example, the total IAA-induced elongation growth of coleoptile segments at 40°C was 1438.1 ± 134.5 µm cm⁻¹ (mean ± SE, n = 11) while elongation of 2747.4 ± 269.7 µm cm⁻¹ (mean ± SE, n = 11) was observed in IAA applied together with FC (Fig. 1A). The data in Figure 1B indicate that coleoptile segments incubated at 40°C (over 2 h), without growth substances (control) characteristically changed the pH of the medium: usually within the first 30–45 min an increase of pH (by ca. 0.5 pH unit) was observed, followed by a slow decrease of pH. When IAA or FC was added (after 2 h of segment''s incubation in control medium), an additional decrease of pH was observed. As can be seen in Figure 1B, FC added at 40°C was much more effective in acidification of the medium, as compared to IAA. For FC, 5h after its addition, the pH of the incubation medium dropped to pH 4.2, whereas for IAA the pH was only 5.4. However, addition of IAA together with FC at 40°C dropped medium pH approximately to the same value as was observed in the presence of FC only.Open in a separate windowFigure 1Effect of high temperature (40°C) on growth (A) and medium pH (B) of maize coleoptile segments incubated in the presence of IAA (10 µM) and FC (1 µM). The growth of a stack of 21 segments, expressed as elongation (µm cm⁻¹), was measured simultaneously with medium pH at 40°C. After preincubation (over 2 h) of the coleoptile segments in control medium, IAA and FC was added (arrow). Values are means of 11 independent experiments. Bars indicate ± SE. In the case of medium pH SE did not exceed 8%.In conclusion, the results presented in this addendum provide further evidence that FC on the receptor level is much more effective than IAA.     

The effect of the seed coat, embryonic axis and aeration conditions on starch degradation hi cotyledons of Lens culinaris

The effect of exogenously applied galactose on the cell wall polysaccharide synthesis and UDP-sugar levels in oat ( Avena sativa L. cv. Victory I) coleoptile segments was studied to clarify the mechanism of inhibition of IAA-induced cell elongation by galactose, and the following results were obtained: (1) The inhibition of IAA-induced cell elongation by galactose became apparent after a 2 h-lag, while the lag was shortened to 1 h when galactose was added to the segments after more than 1 h of IAA application. (2) Galactose inhibited the [¹⁴C]-glucose incorporation into cellulosic and non-cellulosic fractions of the cell wall and the increase in net polysaccharide content in the fractions during long-term incubation. (3) The dominant sugar nucleotide in oat coleoptiles was UDP-glucose (2.1 nmol segment⁻¹). Galactose application caused a remarkable decrease in the UDP-glucose level, accompanying a strong accumulation of galactose-1-phosphate and UDP-galactose. (4) Galactose-1-phosphate competitively inhibited the UTP: a- d -glucose-1-phosphate uridylyltransferase (EC 2.7.7.9) activity of the crude enzyme preparation from oat coleoptiles. From these results we conclude that galactose inhibits the IAA-induced cell elongation by inhibiting the formation of UDP-glucose, which is a key intermediate of cell wall polysaccharide synthesis.     

Cellular responses to naphthoquinones: juglone as a case study

The effects of juglone (JG) on the endogenous growth, growth in the presence of either indoleacetic acid (IAA) or fusicoccin (FC) and on proton extrusion were studied in maize coleoptile segments. In addition, membrane potential changes were also determined at chosen JG concentrations. It was found that JG, when added to the incubation medium, inhibited endogenous growth as well as growth in the presence of either IAA or FC. Simultaneous measurements of growth and external pH indicated that inhibition of either IAA-induced growth or proton extrusion by JG was a linear function of JG concentration. Addition of JG to the control medium caused depolarization of the membrane potential (E_m), value of which was dependent on JG concentration and time after its administration. Hyperpolarization of E_m induced by IAA was suppressed in the presence of JG. It was also found that for coleoptile segments initially preincubated with JG, although subsequently removed, addition of IAA was not effective in the stimulation of growth and medium acidification. Taken together, these results suggest that the mechanism by which JG inhibits the IAA-induced growth of maize coleoptile segments involves inhibition of PM H⁺-ATPase activity.     

Use of a pH-response curve for growth to predict apparent wall pH in elongating segments of maize coleoptiles and sunflower hypocotyls

To determine the relationship between apparent pH of the wall solution and shoot segment elongation, curves for the initial growth rates as a function of pH of the external solution were determined for maize (Zea mays L.) coleoptiles and sunflower (Helianthus annuus L.) hypocotyls and used to predict apparent wall pH in segments responding to indole-3-acetic acid (IAA) and fusicoccin (FC). When a solution having a pH predicted for walls of coleoptile segments responding to IAA was applied to the segments in the presence of IAA, this pH was not maintained. However, when the same was done for coleoptile segments responding to FC, the predicted pH was maintained in the external solution. Sunflower hypocotyl tissue did not maintain the external pH at the predicted value in the presence of either IAA or FC. The results indicate that wall loosening in coleoptiles caused by IAA may not be solely controlled by pH in the wall, yet growth (wall loosening) caused by FC apparently is directly related to wall pH. In sunflower the growth response to neither IAA nor FC appears to be directly correlated with wall pH.     

Regulation of electrogenic proton pumping by auxin and fusicoccin as related to the growth of Avena coleoptiles

The temporal relations between early responses to indoleacetic acid (IAA), proton secretion, hyperpolarization of the membrane potential, and growth change during the incubation of segments of oat (Avena sativa L.) coleoptiles in a low salt medium. When IAA is added after pretreatment of several hours, proton secretion increases after a latency of 7 minutes and reaches its maximum 10 to 15 minutes later. This timing coincides with both the increase in growth of the segments and the hyperpolarization of the membrane potential of parenchyma cells, consistent with the hypothesis that the change in membrane voltage reflects the activity of an electrogenic proton pump. The extent of IAA-induced hyperpolarization is substantially reduced by elevating [KCl]₀, most likely because this increases the passive conductance of the membrane. Neither growth nor proton secretion is affected by high [KCl]₀ (30 millimolar), indicating that neither process is limited by the magnitude of the membrane potential. These results are consistent with the acid growth hypothesis. Following short incubation times, however, IAA-induced hyperpolarization and growth are detected within 10 minutes, while acidification of the medium is delayed for more than 40 minutes. This result is seemingly in conflict with the acid growth hypothesis, but in freshly cut tissue, the pH of the external medium may not reflect the pH of the epidermal cell walls. The temporal coincidence of auxin-induced growth and hyperpolarization suggests that in freshly isolated segments the hyperpolarization is a more sensitive indication of proton secretion than is acidification of the external aqueous environment.     

Role of cell-wall biogenesis in the initiation of auxin-mediated growth in coleoptiles of Zea mays L.

The involvement of cell-wall polymer synthesis in auxin-mediated elongation of coleoptile segments from Zea mays L. was investigated with particular regard to the growth-limiting outer epidermis. There was no effect of indole acetic acid (IAA) on the incorporation of labeled glucose into the major polysaccharide wall fractions (cellulose, hemicellulose) within the first 2 h of IAA-induced growth. 2,6-Dichlorobenzonitrile inhibited cellulose synthesis strongly but had no effect on IAA-induced segment elongation even after a pretreatment period of 24 h, indicating that the growth response is independent of the apposition of new cellulose microfibrils at the epidermal cell wall. The incorporation of labeled leucine into total and cell-wall protein of the epidermis was promoted by IAA during the first 30 min of IAA-induced growth. Inhibition of IAA-induced growth by protein and RNA-synthesis inhibitors (cycloheximide, cordycepin) was accompanied by an inhibition of leucine incorporation into the epidermal cell wall during the first 30 min of induced growth but had no effect on the concomitant incorporation of monosaccharide precursors into the cellulose or hemicellulose fractions of this wall. It is concluded that at least one of the epidermal cell-wall proteins fulfills the criteria for a growth-limiting protein induced by IAA at the onset of the growth response. In contrast, the synthesis of the polysaccharide wall fractions cellulose and hemicellulose, as well as their transport and integration into the growing epidermal wall, appears to be independent of growth-limiting protein and these processes are therefore no part of the mechanism of growth control by IAA.Abbreviations CHI cycloheximide - COR cordycepin - DCB 2,6-dichlorobenzonitrile - GLP growth-limiting protein(s) - IAA indole-3-acetic acid     

Comparison of the outer and inner epidermis : inhibition of auxin-induced elongation of maize coleoptiles by glucan antibodies

Polyclonal antibodies, raised against β-d-glucans prepared from oat (Avena sativa L.) caryopses, cross-reacted specifically with (1→3),(1→4)-β-d-glucans when challenged in a dot blot analysis of related polymers bound to a cellulose thin layer chromatography plate. The antibodies suppressed indoleacetic acid (IAA)-induced elongation of segments from maize (Zea mays L.) coleoptiles when the outer surface was abraded. However, IAA-induced elongation of nonabraded segments or segments with abrasion restricted to the interior of the cylinder was not influenced by the antibodies. Fab fragments prepared from the antibodies gave similar results. The capacity for IAA to overcome outward curvature of split coleoptile segments was partially reversed by treatment of the segments with the antibodies. Fluorescence microscopy revealed that antibody penetration was largely restricted to the epidermal cell wall region. These results support the view that the degradation of (1→3),(1→4)-β-d-glucans in the outer epidermal cell wall serves an essential role in auxin-induced elongation of Poaceae coleoptiles.     

UDP-Glucose level as a limiting factor for IAA-induced cell elongation in Avena coleoptile segments

The effects of galactose on IAA-induced elongation and endogenous level of UDP-glucose (UDPG) in oat ( Avena sativa L. cv. Victory) coleoptile segments were examined under various growth conditions to see if there was a correlation between the level of UDPG and auxin-induced growth. The following results were obtained:

• (1)

  Galactose (10 m M ) inhibited the auxin-induced cell elongation of oat coleoptile segments after a lag of ca 2 h. Determinations of cell wall polysaccharides and UDP-sugars indicated that galactose, when inhibiting the cell wall polysaccharide synthesis, decreased the level of UDPG but caused an increase in the levels of Gal-1-P and UDP-Gal.
• (2)

  When coleoptile segments treated with IAA and galactose were transferred to galactose-free IAA-solution, the segment elongation was restored and the amounts of cell wall polysaccharides increased. During this period, the amount of UDPG increased and the levels of Gal-1-P and UDP-Gal slightly decreased or leveled off. The UDP-pentoses changed similarly as UDPG did.
• (3)

  Addition of sucrose (30 m M ) enhanced IAA-induced cell elongation and removed growth inhibition by 1 m M galactose. Sucrose increased the amounts of the cell wall polysaccharides and the level of UDPG in the presence or absence of IAA and also counteracted the decrease in UDPG caused by galactose.

These results indicate that the level of UDPG is an important limiting factor for cell wall biosynthesis and, thus, for auxin-induced elongation.     

Evidence for Two Indoleacetic Acid-Induced Growth Responses in the Avena Straight-Growth Indoleacetic Acid Assay

Floating Avena sativa L. cv Victory coleoptile segments were used to determine whether the straight-growth indoleacetic acid (IAA) assay can be reconciled with the Avena curvature assay and the Cholodny-Went theory of photo- and gravitropism. Measurements of segment length after 5 h yield sigmoid-shaped IAA dose-response curves with the growth rate leveling off at 1 [mu]M. However, measurements made at 24 h generate bell-shaped curves with maximal growth being induced by 10 [mu]M IAA. The difference between short- and long-term IAA dose-response curves is not due to IAA degradation; instead, it is the result of two growth responses to IAA. The initial one is rapid, responds to low concentrations of IAA, and lasts for 12 h. The second response is less sensitive to IAA than the first one. It appears after 6 h but is not obvious until the last 12 h of a 24-h incubation. The profile of short-term IAA dose-response curves reflects the initial growth response, whereas that of the 24-h curve is the sum of both growth responses. Linear-linear plots of 5- and 24-h dose-response curves show that coleoptile segment growth rate is proportional to IAA concentration up to 0.3 [mu]M. When the efficiency of IAA action is taken into account, it is found that the most effective IAA concentration for short and long incubations is 0.4 [mu]M. It is concluded that the Avena straight-growth IAA assay is as sensitive as the Avena coleoptile curvature assay, and that it is consistent with the Cholodny-Went theory.