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.     

Promotion of stem elongation by indole-3-butyric acid in intact plants of Pisum sativum L.

While indole-3-butyric acid (IBA) has been confirmed to be an endogenous form of auxin in peas, and may occur in the shoot tip in a level higher than that of indole-3-acetic acid (IAA), the physiological significance of IBA in plants remains unclear. Recent evidence suggests that endogenous IAA may play an important role in controlling stem elongation in peas. To analyze the potential contribution of IBA to stem growth we determined the effectiveness of exogenous IBA in stimulating stem elongation in intact light-grown pea seedlings. Aqueous IBA, directly applied to the growing internodes via a cotton wick, was found to be nearly as effective as IAA in inducing stem elongation, even though the action of IBA appeared to be slower than that of IAA. Apically applied IBA was able to stimulate elongation of the subtending internodes, indicating that IBA is transported downwards in the stem tissue. The profiles of growth kinetics and distribution suggest that the basipetal transport of IBA in the intact plant stem is slower than that of IAA. Following withdrawal of an application, the residual effect of IBA in growth stimulation was markedly stronger than that of IAA, which may support the notion that IBA conjugates can be a better source of free auxin through hydrolysis than IAA conjugates. It is suggested that IBA may serve as a physiologically active form of auxin in contributing to stem elongation in intact plants.     

Effect of Gibberellic Acid on Rate of Extension and Maturation of Pea Internodes

Gibberellic acid (GA) does not delay maturation of pea internodes;on the con-trary, maturation (i.e. cessation of extension) takesplace slightly earlier. Thus the increased length of internodesresulting from GA treatment is due entirely to increased rateof extension. In this experiment, GA treatment of plants accelerated the visibleproduction of the first flower bud by about 4 days: the nodebearing the first flower was not altered. The total number offlower buds produced by the end of the experiment was increasedas a result of GA treatment, but many of those first formedon plants receiving high doses (I-IOµg.) withered beforeopening.     

Elongation of Stem Internodes in the Japanese Morning Glory (Pharbitis nil) in Relation to Auxin Destruction

The relationship between auxin destruction and stem internode elongation was investigated in the vines of the Japanese morning glory (Pharbitis nil Choisy). In young plants an age-dependent gradient was demonstrated in which the decreasing rate of elongation of older internodes correlated with an increasing ability of such tissue to destroy indoleacetic acid. Fragments of tissue from old internodes when incubated with indoleacetic acid (IAA), destroyed the hormone immediately and rapidly; in contrast, young, rapidly elongating internode tissue destroyed IAA only after a lag of several hours. In older plants the gradient was more erratic towards the middle of the plant but old and young tissue behaved as in young plants, i.e., old internodes destroyed IAA rapidly whereas young internodes did not. It appears reasonable to conclude that cessation of elongation in maturing internodes is brought about by developing an internal environment in which auxin is rapidly destroyed.     

Effects of 2-Chloroethylphosphonic Acid and Gibberellic Acid on Sex Expression and Growth of Pumpkins

Treatment of pumpkin plants with 2-chloroethylphosphonic acid (CEPHA) induced a greater production of female flowers, shorter internodes and earlier fruit set while treatment with gibberellic acid (GA) induced a greater production of male flowers, longer internodes and later fruit set. Although CEPHA induced the production of a greater number of female flowers, the bulk of the flowers aborted and only a slight increase in the number of fruits per pumpkin plant occurred. The addition of equal concentrations of CEPHA and GA resulted in pumpkin plants with longer internodes and a greater number of female flowers than the untreated plants, although GA partially overcame the effect of CEPHA. The mode of action of CEPHA and GA on sex expression is discussed.     

Plant hormone interactions: how complex are they?

Models describing plant hormone interactions are often complex and web-like. Here we assess several suggested interactions within one experimental system, elongating pea internodes. Results from this system indicate that at least some suggested interactions between auxin, gibberellins (GAs), brassinosteroids (BRs), abscisic acid (ABA) and ethylene do not occur in this system or occur in the reverse direction to that suggested. Furthermore, some of the interactions are relatively weak and may be of little physiological relevance. This is especially true if plant hormones are assumed to show a log-linear response curve as many empirical results suggest. Although there is strong evidence to support some interactions between hormones (e.g. auxin stimulating ethylene and bioactive GA levels), at least some of the web-like complexities do not appear to be justified or are overstated. Simpler and more targeted models may be developed by dissecting out key interactions with major physiological effects.     

Auxin-Gibberellin Interactions and Their Role in Plant Growth

Recently it was discovered that auxin promotes gibberellin (GA) biosynthesis in decapitated stems of pea (Pisum sativum L.) and tobacco (Nicotiana tabacum L.), and here we review the evidence for this interaction. We also discuss the possible relationship between auxin and the mechanisms by which bioactive GAs (such as GA1) regulate their own levels, and the implications of the auxin-GA interaction for the control of plant growth. It is now possible to envisage auxin as a messenger linking the apical bud with the biosynthesis of active GAs in the expanding internodes. Finally, new evidence is presented that the promotion of growth by GA1 does not depend on GA1-induced increases in auxin content.     

Control of Internode Length in Pisum sativum (Further Evidence for the Involvement of Indole-3-Acetic Acid)

The effects of altered endogenous indole-3-acetic (IAA) levels on elongation in garden pea (Pisum sativum L.) plants were investigated. The auxin transport inhibitors 2,3,5-triiodobenzoic acid (TIBA) and 9-hydroxyfluorene-9-carboxylic acid (HFCA) were applied to elongating internodes of wild-type and mutant lkb plants. The lkb mutant was included because elongating lkb internodes contained 2- to 3-fold less free IAA than those of the wild type. In the wild type, TIBA reduced both the IAA level and internode elongation below the site of application. Both TIBA and HFCA strongly promoted the elongation of lkb internodes and also raised IAA levels above the application site. The synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) also markedly increased internode elongation in lkb plants and virtually restored petioles and tendrils to their wild-type length. In contrast, treatment of wild-type plants with TIBA, HFCA, or 2,4-D caused little or no increase in elongation above the application site. The ethylene synthesis inhibitor aminoethoxyvinylglycine also increased stem elongation in lkb plants, and combined application of HFCA and aminoethoxy-vinylglycine restored lkb internodes to the wild-type length. It is concluded that the level of IAA in wild-type internodes is necessary for normal elongation, and that the reduced stature of lkb plants is at least partially attributable to a reduction in free IAA level in this mutant.     

Auxin promotes gibberellin biosynthesis in decapitated tobacco plants

Excision of the apical bud (decapitation) of tobacco (Nicotiana tabacum L.) plants reduced the endogenous levels of indole-3-acetic acid (IAA), gibberellin A20 (GA20), and GA1 (the bioactive GA), in internode tissue below the excision site. Application of IAA to the stump of decapitated plants dramatically increased GA20 content, to a level 3-fold greater than in intact plants. Gibberellin A1 content was also increased by IAA. Decapitation reduced the conversion of [14C]GA19 to [14C]GA20 and of [14C]GA20 to [14C]GA1, and appeared to promote the deactivation pathway [14C]GA20 to [14C]GA29 to [14C]GA29-catabolite. Application of auxin counteracted these effects, but did not restore the conversion of [14C]GA20 to [14C]GA1 to the level found in intact plants. The results indicate that auxin is necessary for normal GA biosynthesis in stems of tobacco.     

Auxin-Gibberellin Interactions in Pea: Integrating the Old with the New

Recent findings on auxin-gibberellin interactions in pea are reviewed, and related to those from studies conducted in the 1950s and 1960s. It is now clear that in elongating internodes, auxin maintains the level of the bioactive gibberellin, GA₁, by promoting GA₁ biosynthesis and by inhibiting GA₁ deactivation. These effects are mediated by changes in expression of key GA biosynthesis and deactivation genes. In particular, auxin promotes the step GA₂₀ to GA₁, catalyzed by a GA 3-oxidase encoded by Mendel’s LE gene. We have used the traditional system of excised stem segments, in which auxin strongly promotes elongation, to investigate the importance for growth of auxin-induced GA₁. After excision, the level of GA₁ in wild-type (LE) stem segments rapidly drops, but the auxin indole-3-acetic acid (IAA) prevents this decrease. The growth response to IAA was greater in internode segments from LE plants than in segments from the le-1 mutant, in which the step GA₂₀ to GA₁ is impaired. These results indicate that, at least in excised segments, auxin partly promotes elongation by increasing the content of GA₁. We also confirm that excised (light-grown) segments require exogenous auxin in order to respond to GA. On the other hand, decapitated internodes typically respond strongly to GA₁ application, despite being auxin-deficient. Finally, unlike the maintenance of GA₁ content by auxin, other known relationships among the growth-promoting hormones auxin, brassinosteroids, and GA do not appear to involve large changes in hormone level.     

Correlations between net auxin and secondary xylem development in young Populus deltoides

Stems of cottonwood ( Populs deltoides Bartr. ex Marsh.) plants grown under different conditions were examined to determine the relation between net endogenous auxin yields and the acropetal advance of the primary-secondary vascular transition zone (TZ). In all treatments, the internode yielding maximum net auxin activity, as determined by the Avena curvature bioassay, closely corresponded with the internode in which the TZ occurred. Under short-day (SD) dormancy-inducing conditions, auxin yield declined steadily while the maximum auxin peak and the TZ shifted toward younger internodes. Auxin yields from these plants were extremely low after 5 weeks of SD compared with those from long-day (LD) plants. The only consistent auxin yield was obtained from internodes subtending young leaves beneath the apical bud. Plants placed in SD for 3 weeks and then returned to LD conditions showed an immediate increase in auxin yield in the stem, and the progressive acropetal advance of the TZ under SD was reversed. Therefore, within 7 LD the positions of the TZ and peak auxin yield corresponded to those observed before the imposition of SD Fully dormant plants placed in LD showed a dramatic rise in auxin yields during the first 2 weeks of renewed growth. Although low levels of auxin were found in the newly developing shoots after 6 LD, yields increased rapidly after 9 and 14 LD. The position of the TZ corresponded with the peak of net auxin activity after 9 and 14 LD.     

REGULATORY ROLE OF INDOLE-3-ACETIC ACID AND GIBBERELLIC ACID IN VEGETATIVE DEVELOPMENT OF XANTHIUM PENNSYLVANICUM

A single application of gibberellic acid to young internodes significantly accelerated the rate of internode growth and the rate of leaf production in shoots of Xanthium pennsylvanicum Wallr. The average duration of one plastochron in treated plants was reduced to 43% of control levels. Gibberellic acid also had a pronounced morphogenetic effect on leaves so that the area and leaf length of treated plants were both significantly reduced. Depending upon concentration, auxin had both inhibitory and promotive effects on Xanthium shoots. Indole-3-acetic acid markedly altered the response of the gibberellic acid-treated internodes and those located above and below the site of application. In addition, high auxin concentrations induced the formation of adventitious roots in treated internodes. Auxin also brought about significant reductions in the length and area of leaves developed under the influence of this hormone.     

Changes in Phytohormone Status in Stems and Roots after Decapitation of Pea Seedlings

Experiments were performed on the first and second internodes and 4-cm-long apical segments of main roots of pea (Pisum sativum L.) seedlings, grown in the light and decapitated above the second node on the seventh day after seed germination. Endogenous phytohormones were measured by the enzyme-linked immunosorbent assay during three days after decapitation of seedlings. The IAA level in the internodes decreased 2–3 times on the second day after decapitation of seedlings while the cytokinin level increased 5–6 times for zeatin and zeatin riboside (Z and ZR) and 1.5–2 times for isopentenyl adenine and isopentenyl adenosine (IP and IPA). In contrast to internodes, the IP and IPA contents in the roots of decapitated seedlings did not change, but the levels of Z and ZR increased 1.5–2 times compared to intact plant roots. The IAA level in the apical region of root remained almost unchanged after the removal of shoot apex. It was concluded that the apical meristem of the main root is not the site of the cytokinin response to the auxin signal coming from the stem apex and that a slight accumulation of Z and ZR after decapitation is due to upper zones of the root. There was no difference in the content of gibberellin-like substances between the internodes of intact and decapitated seedlings. However, the content of gibberellins (GA) in the root tip decreased after decapitation of seedling, which suggests an essential role of apical bud in supplying the root with GA and/or intermediates for their biosynthesis.     

Magnitude and Kinetics of Stem Elongation Induced by Exogenous Indole-3-Acetic Acid in Intact Light-Grown Pea Seedlings

Exogenously applied indole-3-acetic acid (IAA) strongly promoted stem elongation over the long term in intact light-grown seedlings of both dwarf (cv Progress No. 9) and tall (cv Alaska) peas (Pisum sativum L.), with the relative promotion being far greater in dwarf plants. In dwarf seedlings, solutions of IAA (between 10-4 and 10-3 M), when continuously applied to the uppermost two internodes via a cotton wick, increased whole-stem growth by at least 6-fold over the first 24 h. The magnitude of growth promotion correlated with the applied IAA concentration from 10-6 to 10-3 M, particularly over the first 6 h of application. IAA applied only to the apical bud or the uppermost internode of the seedling stimulated a biphasic growth response in the uppermost internode and the immediately lower internode, with the response in the latter being greatly delayed. This demonstrates that exogenous IAA effectively promotes growth as it is transported through intact stems. IAA withdrawal and reapplication at various times enabled the separation of the initial growth response (IGR) and prolonged growth response (PGR) induced by auxin. The IGR was inducible by at least 1 order of magnitude lower IAA concentrations than the PGR, suggesting that the process underlying the IGR is more sensitive to auxin induction. In contrast to the magnitude of the IAA effect in dwarf seedlings, applied IAA only doubled the growth in tall seedlings. These results suggest that endogenous IAA is more growth limiting in dwarf plants than in tall plants, and that auxin promotes stem elongation in the intact plant probably by the same mechanism of action as in isolated stem segments. However, since dwarf plants to which IAA was applied failed to reach the growth rate of tall plants, auxin cannot be the only limiting factor for stem growth in peas.     

PRIMARY PHLOEM REGENERATION WITHOUT CONCOMITANT XYLEM REGENERATION: ITS HORMONE CONTROL IN COLEUS

Phloem regeneration in the absence of xylem regeneration was evoked in number 5 internodes of Coleus blumei Benth. by severing xylemless phloem bundles. Its quantitative extent was estimated. To determine whether phloem regeneration is directly affected by auxin, or whether it is a secondary consequence of the auxin-dependent xylem regeneration which usually accompanies it, phloem regeneration was measured in decapitated plants from which auxin-producing leaves and buds had been removed (i.e., in “plant stumps”). In these stumps, 1% IAA in lanolin completely restored phloem regeneration to the intact plant level. In such stumps from which roots had been excised, and in excised internodes, IAA failed to restore it to this level. However, zeatin or zeatin riboside in aqueous solution applied to the bases of excised internodes receiving IAA at their apical ends restored phloem regeneration to the level of that in whole plants. When similarly tested, other cytokinins (kinetin, kinetin riboside, 2iP, and 2iPA), gibberellic acid (GA₃), glutamine, proline, sucrose, and a mixture of mineral salts failed to promote phloem regeneration. Glutamic acid, tested only once, was slightly promotive of it.     

The Relation between Cell-wall and Protein Synthesis in Dwarf Pea Plants treated with Gibberellic Acid

The amount of protein and soluble nitrogen present in expandinginternodes of intact dwarf pea seedlings, was investigated atdifferent times after treatment of plants with gibberellic acid(GA). There was a marked increase in rate of protein synthesisfollowing GA treatment, but the rate of synthesis did not keeppace with internode expansion, so that the amount of proteinper unit length fell. The rate of cell-wall synthesis was alsoincreased, and, in contrast to protein, the amount of cell wallper unit length remained approximately constant during internodeexpansion. It is suggested that the increased rate of cell-wallsynthesis which follows GA treatment is mediated by a changein protein metabolism. The amount of soluble nitrogen presentin expanding internodes was also increased. There was littleeffect of GA upon the protein content of internodes which werealmost fully expanded at the time of treatment.     

Interactions of auxin and gibberellin in the control of basal growth of <Emphasis Type="Italic">Arabidopsis</Emphasis> rosette leaves

Arabidopsis is a species that naturally displays the rosette form. Therefore, elucidation of the factors, which control basal leaf development, is of particular interest. Most evidence points that auxins and gibberellins are important in the control of rosette leaf development. In this paper, we report on a regimen that disrupts the normal rosette growth in Arabidopsis and induces internodal growth, which we have termed unbasal. The growth conditions are: (1) seed germination in the presence of 2,3,5-triiodobenzoic acid (TIBA); (2) transfer of the seedlings to a medium containing exogenous auxin (NAA) and GA₃; (3) transfer of the seedlings to a GA₃-only medium for all subsequent growth. Under these conditions, auxin and GA interact to induce internode elongation. Polar auxin transport appears to have a temporal effect on this synergistic interaction. In this regimen, GA increases auxin activity in the basal portions of the stem. Cross sectional morphology of the elongated internodes between two rosette leaves in an un-basal plant was similar to that seen for the pin1 Arabidopsis mutation.     

Auxin metabolism in the root apical meristem

Within the root meristem of flowering plants is a group of mitotically inactive cells designated the quiescent center (QC). Recent work links the quiescent state to high levels of the growth regulator auxin that accumulates in the QC via polar transport. This in turn results in elevated levels of the enzyme ascorbic acid oxidase (AAO), resulting in a reduction of ascorbic acid (AA) within the QC and mitotic quiescence. We present evidence for additional interactions between auxin, AAO, and AA, and report that, in vitro, AAO oxidatively decarboxylates auxin, suggesting a mechanism for regulating auxin levels within the QC. We also report that oxidative decarboxylation occurs at the root tip and that an intact root cap must be present for this metabolic event to occur. Finally, we consider how interaction between auxin and AAO may influence root development by regulating the formation of the QC.