The Influence of Auxin and Ethylene on Chromatin-directed Ribonucleic Acid Synthesis in Soybean Hypocotyl

Soybean seedlings treated with ethylene exhibited small increases in ribonucleic acid content in the elongating section of the hypocotyl. Chromatin isolated from the elongating section of ethylene-treated seedlings showed a 35 to 60% increase in the capacity for RNA synthesis. The ethylene-induced response was saturated at 1 microliter/liter of ethylene and was fully expressed after 3 hours. Auxin caused marked accumulation of RNA and DNA in the elongating and basal tissue of the hypocotyl. Chromatin isolated from these auxin-treated tissues showed an 8- to 10- fold increase in RNA synthetic capacity as measured in vitro. Ethylene added with auxin reduced the auxin enhancement of nucleic acid synthesis in the elongating and basal tissues. Both ethylene and auxin treatment of the seedlings inhibited nucleic acid accumulation and chromatin activity in the apical tissue. Ethylene did not appear to mediate the auxin effects on nucleic acid synthesis in soybean hypocotyl with the possible exception of inhibition in the apical tissue.     

Mechanical inhibition of hypocotyl elongation induces radial enlargement: implications for cytokinin action

Excised elongating segments from 3-day-old soybean (Glycine max var. Wayne) seedlings radially enlarged when auxin-promoted elongation was mechanically inhibited. Growth was similar to segments treated with auxin plus cytokinin. This observation suggests that cytokinin does not necessarily directly “reorient” cell enlargement. Cytokinin-induced radial cell growth may be a secondary effect of cytokinin's inhibition of auxin-promoted elongation.     

Cytokinin inhibits a subset of diageotropica-dependent primary auxin responses in tomato

Many aspects of plant development are regulated by antagonistic interactions between the plant hormones auxin and cytokinin, but the molecular mechanisms of this interaction are not understood. To test whether cytokinin controls plant development through inhibiting an early step in the auxin response pathway, we compared the effects of cytokinin with those of the dgt (diageotropica) mutation, which is known to block rapid auxin reactions of tomato (Lycopersicon esculentum) hypocotyls. Long-term cytokinin treatment of wild-type seedlings phenocopied morphological traits of dgt plants such as stunting of root and shoot growth, reduced elongation of internodes, reduced apical dominance, and reduced leaf size and complexity. Cytokinin treatment also inhibited rapid auxin responses in hypocotyl segments: auxin-stimulated elongation, H(+) secretion, and ethylene synthesis were all inhibited by cytokinin in wild-type hypocotyl segments, and thus mimicked the impaired auxin responsiveness found in dgt hypocotyls. However, cytokinin failed to inhibit auxin-induced LeSAUR gene expression, an auxin response that is affected by the dgt mutation. In addition, cytokinin treatment inhibited the auxin induction of only one of two 1-aminocyclopropane-1-carboxylic acid synthase genes that exhibited impaired auxin inducibility in dgt hypocotyls. Thus, cytokinin inhibited a subset of the auxin responses impaired in dgt hypocotyls, suggesting that cytokinin blocks at least one branch of the DGT-dependent auxin response pathway.     

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.     

The hormonal regulation of axillary bud growth in Arabidopsis

Apically derived auxin has long been known to inhibit lateral bud growth, but since it appears not to enter the bud, it has been proposed that its inhibitory effect is mediated by a second messenger. Candidates include the plant hormones ethylene, cytokinin and abscisic acid. We have developed a new assay to study this phenomenon using the model plant Arabidopsis. The assay allows study of the effects of both apical and basal hormone applications on the growth of buds on excised nodal sections. We have shown that apical auxin can inhibit the growth of small buds, but larger buds were found to have lost competence to respond. We have used the assay with nodes from wild-type and hormone-signalling mutants to test the role of ethylene, cytokinin and abscisic acid in bud inhibition by apical auxin. Our data eliminate ethylene as a second messenger for auxin-mediated bud inhibition. Similarly, abscisic acid signalling is not to be required for auxin action, although basally applied abscisic can enhance inhibition by apical auxin and apically applied abscisic acid can reduce it. By contrast, basally applied cytokinin was found to release lateral buds from inhibition by apical auxin, while apically applied cytokinin dramatically increased the duration of inhibition. These results are consistent with cytokinin acting independently to regulate bud growth, rather than as a second messenger for auxin. However, in the absence of cytokinin-signalling mutants, a role for cytokinin as a second messenger for auxin cannot be ruled out.     

Growth and stomata development of Arabidopsis hypocotyls are controlled by gibberellins and modulated by ethylene and auxins

The plant hormones gibberellin (GA), ethylene and auxin can promote hypocotyl elongation of Arabidopsis seedlings grown in the light on a low nutrient medium (LNM). In this study, we used hypocotyl elongation as a system to investigate interactions between GA and ethylene or auxin and analysed their influence on the development of stomata in the hypocotyl. When applied together, GA and ethylene or auxin exerted a synergistic effect on hypocotyl elongation. Stimulated cell elongation is the main cause of hypocotyl elongation. Furthermore, hypocotyls treated with GA plus either ethylene or auxin show an increased endoreduplication. In addition, a small but significant increase in cell number was observed in the cortical cell files of hypocotyls treated with ethylene and GA together. However, studies with transgenic seedlings expressing CycB1::uidA genes revealed that cell division in the hypocotyl occurs only in the epidermis and mainly to form stomata, a process strictly regulated by hormones. Stomata formation in the hypocotyl is induced by the treatment with either GA or ethylene. The effect of GA could be strongly enhanced by the simultaneous addition of ethylene or auxin to the growth medium. Gibberellin is the main signal inducing stomata formation in the hypocotyl. In addition, this signal regulates hypocotyl elongation and is modulated by ethylene and auxin. The implication of these three hormones in relation to cell division and stomata formation is discussed.     

Correlation of Endogenous Gibberellic Acid with Initiation of Mango Shoot Growth

Stems of mango (Mangifera indica L.) rest in a nongrowing, dormant state for much of the year. Ephemeral flushes of vegetative or reproductive shoot growth are periodically evoked in apical or lateral buds of these resting stems. The initiation of shoot growth is postulated to be primarily regulated by a critical ratio of root-produced cytokinins, which accumulate in buds and by leaf-produced auxin, which decreases in synthesis and transport over time. Exogenously applied gibberellic acid (GA3) delays initiation of bud break but does not determine whether the resulting flush of growth is vegetative or reproductive. We tested the hypothesis that endogenous GA3, which influences release of these resting buds, may decrease in stem tips or leaves with increasing age of mango stems. GA3 and several other GAs in stem tip buds and leaves were identified and quantified in stems of different ages. The major endogenous GAs found in apical buds and leaves of vegetative mango stems were early 13-hydroxylation pathway gibberellins: GA1, epi-GA1, GA3, GA19, GA20, and GA29, as identified by gas chromatography-mass spectrometry (GC-MS). A novel but unidentified GA-like compound was also present. The most abundant GAs in apical stem buds were GA3 and GA19. Contrary to the hypothesis, the concentration of GA3 increased within buds with increasing age of the stems. The concentrations of other GAs in buds were variable. The concentration of GA3 did not change significantly with age in leaves, whereas that of most of the other GAs declined. GA1 levels were greatest in leaves of elongating shoots. These results are consistent with the concept that rapid shoot growth is associated with synthesis of GAs leading to GA1. The role of GA3 in delaying bud break in mango is not known, but it is proposed that it may enhance or maintain the synthesis or activity of endogenous auxin. It, thereby, maintains a high auxin/cytokinin ratio similar to responses to GA3 that maintain apical dominance in other plant species.     

Correlation of Endogenous Gibberellic Acid with Initiation of Mango Shoot Growth

Stems of mango (Mangifera indica L.) rest in a nongrowing, dormant state for much of the year. Ephemeral flushes of vegetative or reproductive shoot growth are periodically evoked in apical or lateral buds of these resting stems. The initiation of shoot growth is postulated to be primarily regulated by a critical ratio of root-produced cytokinins, which accumulate in buds and by leaf-produced auxin, which decreases in synthesis and transport over time. Exogenously applied gibberellic acid (GA3) delays initiation of bud break but does not determine whether the resulting flush of growth is vegetative or reproductive. We tested the hypothesis that endogenous GA3, which influences release of these resting buds, may decrease in stem tips or leaves with increasing age of mango stems. GA3 and several other GAs in stem tip buds and leaves were identified and quantified in stems of different ages. The major endogenous GAs found in apical buds and leaves of vegetative mango stems were early 13-hydroxylation pathway gibberellins: GA1, epi-GA1, GA3, GA19, GA20, and GA29, as identified by gas chromatography-mass spectrometry (GC-MS). A novel but unidentified GA-like compound was also present. The most abundant GAs in apical stem buds were GA3 and GA19. Contrary to the hypothesis, the concentration of GA3 increased within buds with increasing age of the stems. The concentrations of other GAs in buds were variable. The concentration of GA3 did not change significantly with age in leaves, whereas that of most of the other GAs declined. GA1 levels were greatest in leaves of elongating shoots. These results are consistent with the concept that rapid shoot growth is associated with synthesis of GAs leading to GA1. The role of GA3 in delaying bud break in mango is not known, but it is proposed that it may enhance or maintain the synthesis or activity of endogenous auxin. It, thereby, maintains a high auxin/cytokinin ratio similar to responses to GA3 that maintain apical dominance in other plant species.     

The role of ethylene in 2.4-D-induced growth inhibition

Summary Ethylene and 2.4-dichlorophenoxyacetic acid (2.4-D) inhibited the growth of etiolated soybean (Glycine max cv. Hawkeye) seedlings causing tissue swelling and an increase in RNA, DNA and protein content in the subapical hypocotyl tissue. 2.4-D increased ethylene evolution from soybean seedlings and it was found that some of the effect of this herbicide on soybeans was due to the increased ethylene production.Ethylene is responsible in part for the inhibition of elongation and of increase in weight that occurs at supraoptimal concentrations of 2.4-D applied to excised hypocotyl sections. Abscisic acid inhibits 2.4-D-induced tissue swelling and ethylene production in the excised, elongating section. The cotyledons of the soybean seedlings appear to regulate the 2.4-D-induced production of ethylene and the roots are necessary for the 2.4-D-induced tissue swelling.     

Uncoupling Auxin and Ethylene Effects in Transgenic Tobacco and Arabidopsis Plants

Overproduction of auxin in transgenic plants also results in the overproduction of ethylene. Plants overproducing both auxin and ethylene display inhibition of stem elongation and growth, increased apical dominance, and leaf epinasty. To determine the relative roles of auxin and ethylene in these processes, transgenic tobacco and Arabidopsis plants expressing the auxin-overproducing tryptophan monooxygenase transgene were crossed to plants expressing an ethylene synthesis-inhibiting 1-aminocyclopropane-1-carboxylate deaminase transgene. Tobacco and Arabidopsis plants with elevated auxin and normal levels of ethylene were obtained by this strategy. Transgenic auxin-overproducing Arabidopsis plants were also crossed with the ethylene-insensitive ein1 and ein2 mutants. Analysis of these plants indicates that apical dominance and leaf epinasty are primarily controlled by auxin rather than ethylene. However, ethylene is partially responsible for the inhibition of stem elongation observed in auxin-overproducing tobacco. Finally, these data show that auxin overproduction can be effectively uncoupled from ethylene overproduction in transgenic plants to enable direct manipulation of plant morphology for agronomic and horticultural purposes.     

Effect of Competition and Treatment with Inhibitor of Ethylene Perception on Growth and Hormone Content of Lettuce Plants

We elucidated the effect of increased planting density (single and grouped competing plants) on concentrations of auxin, abscisic acid, and cytokinins in normal lettuce plants and in those with ethylene perception inhibited by 1-methylcyclopropene (1-MCP). An attempt was made to relate the changes in hormone concentration induced by competition and inhibition of ethylene sensitivity to growth responses of lettuce planting. The results showed changes in concentrations of auxins, cytokinins, and ABA in the response of lettuce to crowding. Accumulation of ABA in shoots was likely to contribute to inhibition of transpiration of the plants grown in the presence of neighbors. This assumption was supported by the results of application of an inhibitor of ABA synthesis (fluridone and carotenoid biosynthesis herbicide) resulting in increased transpiration of grouped, but not single plants. Increased planting density led to the decline in root auxins paralleled by inhibition of root growth. This effect was likely to be due to decreased auxin transport to the roots from the shoots suggested by accumulation of auxins in the shoots and inhibition of root growth by application of the auxin transport inhibitor [N-(1-naphtyl)phtalamic acid (NPA)]. Importance of the changes in hormone concentrations was confirmed by data showing that disturbance of auxin and cytokinin distribution detected in MCP-treated plants was accompanied by corresponding modification of the growth response.

     

The gravity-regulated growth of axillary buds is mediated by a mechanism different from decapitation-induced release

When the upper part of the main shoot of the Japanese morning glory (Pharbitis nil or Ipomoea nil) is bent down, the axillary bud situated on the uppermost node of the bending region is released from apical dominance and elongates. Here, we demonstrate that this release of axillary buds from apical dominance is gravity regulated. We utilized two agravitropic mutants of morning glory defective in gravisensing cell differentiation, weeping (we) and weeping2 (we2). Bending the main shoots of either we or we2 plants resulted in minimal elongation of their axillary buds. This aberration was genetically linked to the agravitropism phenotype of the mutants, which implied that shoot bending-induced release from apical dominance required gravisensing cells. Previous studies have shown that basipetal translocation of auxin from the apical bud inhibits axillary bud growth, whereas cytokinin promotes axillary bud outgrowth. We therefore compared the roles of auxin and cytokinin in bending- or decapitation-induced axillary bud growth. In the wild-type and we plants, decapitation increased cytokinin levels and reduced auxin response. In contrast, shoot bending did not cause significant changes in either cytokinin level or auxin response, suggesting that the mechanisms underlying gravity- and decapitation-regulated release from apical dominance are distinct and unique.     

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.     

The role of auxin in growth of the plumular hook section of etiolated pea seedling

Externally applied GA greatly promoted elongation of the plumularhook section of the etiolated Alaska pea seedling, but IAA hadno such effect when given either alone or with GA. PCIB inhibitedelongation of the plumular hook section both in the presenceand absence of applied GA. The PCIB effect in the absence ofGA was partially overcome by IAA, but not completely. On theother hand, the PCIB effect in the presence of GA was completelyovercome by IAA. No antagonic response was, however, obtainedbetween GA and PCIB. CCC also retarded elongation of the sectionand this inhibition was completely overcome by GA, but not byIAA. There was little difference in the amount of endogenous auxindetectable in GA treated and untreated sections. These resultssuggest that auxin is necessary for the growth of both GA treatedand untreated plumular hook sections and that auxin and gibberellinact differently on the growth of the section. (Received April 24, 1968; )     

Long-term inhibition by auxin of leaf blade expansion in bean and Arabidopsis

The role of auxin in controlling leaf expansion remains unclear. Experimental increases to normal auxin levels in expanding leaves have shown conflicting results, with both increases and decreases in leaf growth having been measured. Therefore, the effects of both auxin application and adjustment of endogenous leaf auxin levels on midrib elongation and final leaf size (fresh weight and area) were examined in attached primary monofoliate leaves of the common bean (Phaseolus vulgaris) and in early Arabidopsis rosette leaves. Aqueous auxin application inhibited long-term leaf blade elongation. Bean leaves, initially 40 to 50 mm in length, treated once with alpha-naphthalene acetic acid (1.0 mm), were, after 6 d, approximately 80% the length and weight of controls. When applied at 1.0 and 0.1 mm, alpha-naphthalene acetic acid significantly inhibited long-term leaf growth. The weak auxin, beta-naphthalene acetic acid, was effective at 1.0 mm; and a weak acid control, benzoic acid, was ineffective. Indole-3-acetic acid (1 microm, 10 microm, 0.1 mm, and 1 mm) required daily application to be effective at any concentration. Application of the auxin transport inhibitor, 1-N-naphthylphthalamic acid (1% [w/w] in lanolin), to petioles also inhibited long-term leaf growth. This treatment also was found to lead to a sustained elevation of leaf free indole-3-acetic acid content relative to untreated control leaves. Auxin-induced inhibition of leaf growth appeared not to be mediated by auxin-induced ethylene synthesis because growth inhibition was not rescued by inhibition of ethylene synthesis. Also, petiole treatment of Arabidopsis with 1-N-naphthylphthalamic acid similarly inhibited leaf growth of both wild-type plants and ethylene-insensitive ein4 mutants.     

Red Light-inhibited Mesocotyl Elongation in Maize Seedlings: I. The Auxin Hypothesis

Red light-inhibited mesocotyl elongation, which occurs in intact Zea mays L. seedlings, was studied in excised segments which included the coleoptile (or parts therefrom) and apical centimeter of the mesocotyl. Experiments took into account, first, the ability of the segments to regenerate auxin supply sites, and, second, that auxin uptake can be greatly reduced if there is no cut surface, apical to the elongating cells, to act as a port of entry. In all cases, auxin completely reversed the inhibition of elongation by light. The results support the hypothesis that light regulates mesocotyl elongation by controlling auxin supply from the coleoptile. Sucrose concentration had no effect on auxin reversal of light-inhibited elongation, but relatively high concentrations of gibberellic acid (10 μm) could substitute for auxin in this system.     

The influence of gibberellic acid and abscisic acid on cell and tissue differentiation of bean callus.

Bean callus was induced to form roots (tissue differentiation) and vascular nodules (cell differentiation) by lowering the ratio of auxin to cytokinin in the growth medium. Both types of differentiation were inhibited by the addition of abscisic acid (at concentrations greater than I muM) to induction medium. Initiation of differentiation was inhibited, but its subsequent development was not, and the inhibition was not affected by the addition of gibberellic acid. Addition of gibberellic acid (GA) alone to induction medium stimulated tissue differentiation, although cell differentiation was unaffected (30 muM GA) or inhibited (45 muM GA) and its onset was delayed at both concentrations. Root initiation was also stimulated by gibberellic acid (0.I-45 muM) at an auxin-to-kinin ratio 10 times that normally optimal for cell differentiation. The phenylalanine ammonia lyase (PAL) activity of the calluses was closely correlated with the amount of cell differentiation which had occurred, and measurement of this confirmed that gibberellic acid delayed the initiation of cell differentiation. The increase and subsequent decline of PAL and betaI leads to 3 glucan synthetase activities, normally induced by transfer to induction medium, was abolished by abscisic acid. Addition of gibberellic acid did not affect the betaI leads to 3 glucan synthetase activity.     

Exposure of Petunia Seedlings to Ethylene Decreased Apical Dominance by Reducing the Ratio of Auxin to Cytokinin

Seedlings of Petunia x hybrida ‘Orchid’ treated with the ethylene-releasing compound ethephon at 0.9, 1.7, and 3.5 mM evolved ethylene at a higher rate as the concentration of ethephon increased. Regardless of the concentration of ethephon applied, ethylene evolution peaked 6 to 8 h following application. Evidence that ethephon application decreased apical dominance included an increase in the number of new nodes on the main stem and a sustained increase in the length of new and existing lateral shoots compared to the control (no ethephon). Plants treated with 3.5 mM ethephon developed mild chlorosis, whereas a concentration of 1.7 mM ethephon decreased apical dominance without phytotoxic effects. The auxin/cytokinin ratio decreased in the apical shoot section as early as 1 h after ethephon treatment. In contrast, a decrease in the ratio in the subapical shoot section was not detected until 24 h after ethephon application. Reduction in auxin/cytokinin ratio was a result of a decrease in indole-3-acetic acid (IAA) and an increase of zeatin riboside (ZR), but not isopentenyladenosine (iPA). These results suggest that exposing ‘Orchid’ petunia seedlings to ethylene via ethephon lowers the auxin/cytokinin ratio, thereby promoting the outgrowth of lateral shoots.     

Interaction of ethylene and a cytokinin in promoting hypocotyl elongation in a dwarf strain of watermelon

Experiments were conducted to study the interaction of ethylene and the cytokinin N⁶-benzyladenine (BA) in promoting hypocotyl elongation in a dwarf strain of watermelon (Citrullus lanatus [Thunb] Matsu. and Nakai). Optimum promotion of hypocotyl elongation is elicited by an apical treatment with 0.2 microgram BA. At dosages above 0.3 microgram per apex, BA-enhancement of elongation is reduced concomitant with stimulation of ethylene production and lateral expansion of hypocotyls. Application of the ethylene biogenesis inhibitor, aminoethoxyvinylglycine, at dosages from 0.3 to 10 micrograms per apex inhibited BA-induced ethylene production. In seedlings treated with 0.2 microgram BA, 10 micrograms aminoethoxyvinylglycine per apex reduced ethylene production to about one-third of control levels and reduced BA stimulation of hypocotyl elongation by 74%. Exposure of watermelon seedlings to 60 ± 10 nanoliters per liter of ethylene in a flowing system nearly eliminated aminoethoxyvinylglycine inhibition of BA-promoted growth. The results suggest that physiological levels of internal ethylene are required for cytokinin promotion of hypocotyl elongation in watermelon.