New methods to analyse auxin-induced growth II: The swelling reaction of protoplasts – a model system for the analysis of auxin signal transduction?

A method for monitoring the time course of auxin-induced volume changesby protoplasts at a high temporal resolution was developed for Zeamays coleoptile protoplasts. Auxins, like indole-3-acetic acid(IAA), induce a rapid change in volume. Immediately after addition ofthis auxin, a transient shrinkage was observed, followed by a long-termswelling response. This reaction occurred in the same time window as thetypical auxin growth response of intact coleoptiles. Active auxins, like1-naphthalene acetic acid (1-NAA) and 4-chloroindole-3-acetic acid(4-Cl-IAA), caused similar volume changes, whereas the inactive analogue2-naphthalene acetic acid (2-NAA) had no effect. The phytotoxinfusicoccin (FC) induced a rapid swelling response. We conclude that thissingle cell system is very adequate to analyse mechanisms of auxinsignal transduction.     

Role of Cell Wall-degrading Enzymes in Auxin-induced Cell Expansion in Yeast

Using auxin-responsive diploid strains of Saccharomyces cerevisiae and S. ellipsoideus, we studied the role of cell wall-degrading enzymes in the auxin-induced cell expansion. Highly purified fungal β-l,3-glucanase added to cell suspension caused cell expansion in these strains. The cell expansion induced by auxin was inhibited by the addition of õ-glucono-lactone, which inhibited the activity of a crude β-l,3-gluca-nase preparation from yeast in a competitive manner. Laminarinase activity was significantly higher in the extract from auxin-treated yeast cells than in the extract of cells not treated with auxin. These results support the idea that auxin induces expansion of yeast cells of certain strains through enhancement of the activity of wall polysaccharide-degrading enzymes.     

THE RELATIONSHIP BETWEEN THE PROMOTION OF ELONGATION OF FERN PROTONEMATA BY LIGHT AND GROWTH SUBSTANCES

Germinating spores of the fern Onoclea sensibilis L. were grown in darkness, so that they developed as filaments (protonemata). Brief daily exposure of the filaments to red, far-red or blue light increased the rate of filament elongation. Filament elongation was also promoted by indoleacetic acid. When filament elongation was promoted with both indoleacetic acid and exposure to light, the growth promotions caused by red and far-red light were additive to auxin-induced growth. Blue light promoted elongation only at sub-optimal concentrations of auxin. Elongation induced by guanine was additive to red- and far-red-induced elongation. Gibberellic acid had no effect on elongation under any condition. Blue-light-induced elongation resembled auxin-induced elongation in its requirement for exogenous sucrose and sensitivity to inhibition by parachlorophenoxyisobutyric acid. Red and far-red light were active regardless of the presence or absence of sucrose and promoted elongation at a concentration of parachlorophenoxyisobutyric acid which completely inhibited blue-light-induced elongation.     

The<Emphasis Type="Italic"> diageotropica</Emphasis> mutation of tomato disrupts a signalling chain using extracellular auxin binding protein 1 as a receptor

The diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) is known to lack a number of typical auxin responses. Here we show that rapid auxin-induced growth of seedling hypocotyls is completely abolished by the mutation over the full range of auxin concentrations tested, and also in early phases of the time course. Protoplasts isolated from wild-type hypocotyls respond to auxin by a rapid increase in cell volume, which we measured by image analysis at a high temporal resolution. A similar swelling could be triggered by antibodies directed against a part of the putative auxin-binding domain (box-a) of the auxin-binding protein 1 (ABP1). Induction of swelling both by auxin and by the antibody was not observed in the protoplasts isolated from the dgt mutant. However, dgt protoplasts are able to respond to the stimulator of the H⁺-ATPase, fusicoccin, with normal swelling. We propose that dgt is a signal-transduction mutation interfering with an auxin-signalling pathway that uses ABP1 as a receptor.Abbreviations ABP auxin-binding protein - CCD charge-coupled device - 2,4-D 2,4-dichlorophenoxyacetic acid - dgt diageotropica - FC fusicoccin     

A Requirement for DNA Synthesis during Auxin Induction of Cell Enlargement in Tobacco Pith Tissue

IAA (indoleacetic acid) is known to induce cell enlargement without cell division in tobacco pith explants grown on an agar medium without added cytokinin. The very long lag period before IAA (2 × 10^?5M) stimulates growth, about 3 days, can be useful to study the metabolic changes which lead to the promotion of growth. When the disks are transferred to a medium without IAA after 2 days or less of treatment with IAA, the IAA does not stimulate growth. Disks transferred after 3 days, subsequently show an auxin response, almost as great as those given IAA continuously. At 5 × 10^?4M, 5-fluorodeoxyuridine (FUDR), which inhibits DNA synthesis by blocking formation of thymidylate, completely suppresses the lAA-induced growth if it is added together with the IAA or 1 day later. When the FUDR is given 2 days after the IAA, there is a small increment of auxin-induced growth, and an even greater amount if added after 3 days. The period when exogenous auxin must be present to stimulate growth corresponds to the period of FUDR sensitivity. The FUDR inhibition is prevented by thymidine but not by uridine. Other inhibitors of DNA synthesis, hydroxyurea and fluorouracil, also inhibit auxin-induced growth. Thus DNA synthesis seems to be required for auxin induction of cell enlargement in tobacco pith explants. In contrast, FUDR does not inhibit auxin-induced growth in corn coleoptile and artichoke tuber sections.     

Auxin-induced longitudinal-to-transverse reorientation of cortical microtubules in nonelongating epidermal cells of azuki bean epicotyls

Summary In epidermal cells of azuki bean (Vigna angularis) epicotyl segments, that were sequentially treated with an auxin-free solution and an auxin solution, cortical microtubules changed their orientation from longitudinal to transverse. Auxin caused the reorientation of microtubules from longitudinal to transverse in segments that were kept under anaerobic conditions and, therefore, showed no elongation, indicating that auxin can regulate the orientation of microtubules by a mechanism that does not involve auxin-induced change in the rate of cell elongation.Abbreviations DMSO dimethylsulfoxide - GA₃ gibberellic acid - IAA indoleacetic acid - MT microtubule - PBS phosphate-buffered saline     

PLANT GROWTH AND DEVELOPMENT IN MOLECULAR PERSPECTIVE

• 1 After some false starts in which inactive plant substances were isolated, the isolation and identification of auxin as the growth substance at the meristems and of ethylene as the ripening agent in climacteric fruits represented outstanding achievements.
• 2 In early work, the non-localized origin of auxin at the meristem and its possible transport for coleoptile development were obscured by the superimposition on the results of physiological experiments of the idea of a close parallelism between the plant-growth substances and mammalian hormones. At that time, an absence of chemical instrumentation, suitable for measurement of the tissue levels, compounded the difficulty in interpreting available physiological evidence.
• 3 Member(s) of each of the five groups of naturally occurring plant-growth substances, namely the auxins, cytokinins, gibberellins, ethylene and the growth inhibitors, including abscisic acid, are biologically active at a concentration of 10 μm or less, however, and in this respect they would appear to qualify as candidate phytohormones.
• 4 The sensitivity of plant cells to phytohormones contributes to plant growth and development, and both the variations in sensitivity, for example, of wheat coleoptiles towards growth and the growth of the coleoptiles per se give parallel unimodal relationships with regard to time; the curve representing sensitivity precedes that for growth. A new graphical analysis implies that the growth sensitivity and growth rate functions are mutually interdependent.
• 5 The assumption is made in point 4 that growth substance complexes with receptor protein in growth-sensitive cells, and the concept of receptors would provide explanation for the obvious amplification of effects induced by growth substances.
• 6 Numerous biological situations occur in which the presence of significant amounts of plant hormone controls growth and development. In gravitropism and phototropism, tropistic curvature depends on the difference in physiological concentration of auxin on the two sides of the organ concerned. In infected tobacco plants, the cytokinin to auxin ratios for the tumours determine the kind of development (tumours and shoots, tumours only or tumours plus roots), which takes place.
• 7 Auxin-binding protein has been identified immunologically, and isolated. Work with hormone receptors for gibberellin does not afford unequivocal evidence for more than one primary site of action. Hitherto, no specific receptor protein is known for cytokinins.
• 8 Clear evidence derives, both from structure–activity relationships and from unimodal concentration–response curves, for receptor specificity to auxin action. There is also evidence for a structure–activity relationship in respect of the cytokinin series of compounds.
• 9 From the evidence (points 1–8), there emerges a picture of hormone-induced growth and development of plant cells, which have been made sensitive to hormone through the presence of specific receptor proteins.
• 10 That plant growth and developmental processes involve changes in gene expression would seem to follow from the totipotent nature of meristematic cells, which are capable of specialization in response to phytohormones.
• 11 Auxin regulates de novo synthesis of mRNAs encoding polypeptides essential to the auxin-induced early process of cell elongation. In fact, auxin regulates the concentrations of several authenticated mRNAs and proteins, for example, in elongating soyabean hypocotyl sections.
• 12 Furthermore, two cDNA clones, termed pJCW₁ and pJCW₂ have been isolated with the properties expected of mRNAs involved in the rate-limiting stage of cell elongation. The evidence suggests that the change in relative abundance of the JCW₁ and JCW₂ RNAs is an obligatory auxin-dependent response. Hence, the action of cytokinin in auxin-induced cell elongation would seem to be concerned with the inhibition of rate-limiting proteins, and in fact cytokinin inhibits protein synthesis in excised soyabean hypocotyl.
• 13 Biosystemic experiments on some rapid effects of synthetic auxin growth regulators on mRNA levels in vitro show that there is only partial similarity between those found in pea and soyabean spp. (Leguminosae).
• 14 Two identified sequences, namely TGATAAAAG and GGCAGCATGCA, of two auxin-regulated soyabean genes afford a means for determining whether the auxin-regulation of expression of these genes involves trans-acting regulatory factors.
• 15 The obligatory auxin-induced responses with regard to cell elongation and growth (q.v.) would seem to precede the somewhat mechanical growth properties by which auxin receptive cells secrete H⁺ and lower the pH to yield increased cell-wall plasticity.
• 16 In vertically oriented soyabean seedlings, auxin-regulated RNAs are distributed symmetrically in the elongating region of the hypocotyl, whereas in horizontally oriented seedlings the distribution becomes asymmetrical within a few minutes of horizontal gravitational stimulation. The dynamic expression of auxin-regulated genes is related to the morphogenetic response, initiated by re-distribution of endogenous auxin (point 6).
• 17 In the germination of seeds, the mobilization of food reserves requires hydrolytic enzymes and, in barley grains, gibberellic acid induces de novo biosynthesis of α-amylase and protease. The genetic implications are discussed, and the requirement of dicotyledonous and gymnospermous seeds for the presence of gibberellins is explored.
• 18 In ripening climacteric fruits, ethylene-induced change(s) in gene expression cause de novo biosynthesis of polygalacturonase, which degrades the cell-wall pectin fraction.
• 19 Accordingly, incontestible evidence has been mustered for the proposition that hormone-regulated plant growth and development involves hormone-regulated gene expression.
• 20 As well as the phytohormones, certain environmental factors, such as white light and stress (including anaerobiosis, chilling, heat shock, heavy metal exposure, u.v. light and wounding) have the capacity to regulate gene expression in plants at important stages in growth and development. Discussion at the genetic level focuses on changes produced by:
• 21 The synthesis of phytohormones is significant. For example, as u.v. light-induced regulation of genes produces enzymes for auxin synthesis, it may be responsible in seeds for the endospermal generation of auxins, concerned with the epicotyl/hypocotyl growth in the seedlings.
• 22 Hormones and certain environemental factors (q.v.) initiate some of the numerous stages in plant growth and development, but the regulatory factors are obscure in some other biological situations, such as:
• 23 Utilization of an appropriately re-constituted plant DNA polymerase i in vitro system might enable the type and frequency of misincorporation, produced by plant-growth factors, to be studied. Base-pair substitution changes were produced in strains of crop plant, made resistant to a specific herbicide by genetic engineering (see Hathway, 1989). It is feasible that auxin may behave as a reagent in the chemical sense to effect intramolecular change(s) in some of the sequences concerned, leading to the frame-shift changes observed (see Ainley et al., 1988).

     

On the Relation Between the Effects of Auxin on Growth, pH and Potassium Transport

The relation between the effects of auxin on growth, pH and potassium transport in hypocotyl segments of Helianthus annuus was studied. In a solution containing 20 mM Na₂SO₄ auxin-induced growth was accompanied by an auxin-induced pH drop in the medium. (NH₄)₂SO₄, at the same concentration, brought about an almost complete abolishment of the effect of auxin on the pH. The magnitude of auxin-induced growth was, however, only slightly reduced. This result does not confirm the hypothesis according to which the action of auxin on growth is a result of its effect on the pH. In a solution containing 2 mM sodium phosphate buffer an inhibitory action of IAA on the release of potassium from the tissue was observed. Addition of 20 mM Na₂SO₄ to the medium brought about a complete abolishment of this effect. The magnitude of auxin induced pH drop was, however, similar in the two treatments. It was concluded that, although under suitable experimental conditions, a close relationship may exist between the effects of auxin on pH on K⁺ transport, the coupling between the two phenomena is not obligatory.     

Studies on the role of RNA synthesis in auxin induction of cell enlargement

Selective inhibitors were used to study the connection between nucleic acid synthesis and indoleacetic acid (IAA) induction of cell enlargement. Actinomycin D (act D) and azaguanine (azaG) almost completely inhibit IAA-induced growth in aged artichoke tuber disks when they are added simultaneously with IAA. In contrast, when they are added 24 hours after the hormone, these inhibitors have little or no effect on the induced growth which continues for 48 hours or more with little or no inhibition. Inhibitors of protein synthesis still stop growth when applied 24 hours after the IAA, thus protein synthesis and presumably supporting metabolism are still essential.

In corn coleoptile sections auxin-induced growth did not show any pronounced tendency to become less sensitive to act D as the IAA treatment progressed. Act D did not completely inhibit the response to IAA unless the sections were pretreated with act D for 6 hours. In contrast to act D, cordycepin produced almost complete inhibition of IAA-induced growth when added with the IAA.

Although IAA has a very large and very rapid stimulatory effect (within 10 min) on incorporation of ³²P-orthophosphate into RNA in disks, it did not cause a detectable change in the base composition of the RNA synthesized. Furthermore, the promotive effect could be accounted for through increased uptake of the ³²P. That much of the RNA synthesis in these tissues is not necessary for auxin action is indicated by the results with fluorouracil (FU). FU strongly inhibits RNA synthesis, probably acting preferentially on ribosomal RNA synthesis, without inhibiting auxin-induced growth in the disks or coleoptile sections. FU also strongly inhibited respiration in auxin-treated disks indicating that the large promotion of respiration by auxin likewise may not be entirely necessary for growth.

At least in the artichoke disks, RNA synthesis is required for auxin induction of cell enlargement and not for cell enlargement itself.

The possible relationships of auxin induction of cell enlargement and RNA synthesis are discussed.

     

Auxin-induced growth of tuber tissue of Jerusalem artichoke VIII. Role of cyclic AMP in the action of auxin, cytokinin and gibberellic acid

Cyclic AMP showed no growth-promoting effect when given aloneto unaged slices excised from Jerusalem artichoke tubers. Butit synergistically enhanced cell expansion when given togetherwith an auxin, 2,4-dichlorophenoxyacetic acid. Growth responsesof aged slices to cyclic AMP were much smaller than those ofunaged slices. Cyclic AMP was substantially effective when sliceswere aged in the presence of cyclic AMP, then were transferredto a growth solution containing auxin. Interactions betweencyclic AMP and gibberellic acid or kinetin were additive inpromoting auxininduced cell expansion. Both caffeine and theophylline,inhibitors of phosphodiesterase, enhanced the stimulating effectof applied cyclic AMP on auxin-induced cell expansion. But theydid not enhance the promoting effect of gibberellic acid orkinetin on auxininduced cell expansion. These results suggestthat cyclic AMP did not act as a second messenger for any auxin,gibberellic acid and cytokinin in the promotion of cell expansionin this tissue. (Received September 27, 1972; )     

Rapid auxin-induced root growth inhibition requires the TIR and AFB auxin receptors

We investigated the relation between auxin-induced gene expression and the rapid auxin-induced growth inhibition in Arabidopsis thaliana roots. The natural auxin indole-3-acetic acid (IAA) induced a strong activation of gene expression as visualized by the DR5rev::GFP reporter gene technique. This effect was specific for active auxins and was abolished in knockout mutants of the F-box auxin receptors. We measured the IAA-induced growth inhibition at high time resolution and show that the F-box auxin receptor mutants failed to display this effect. We conclude that the F-box auxin receptors are needed for the response. In hypocotyls, auxin induces an increase in elongation growth, and this effect has been earlier shown to be independent of the F-box receptors. Based on these findings, we discuss differences in the growth control modes in roots and shoots. We demonstrate that the rapid auxin-induced root growth inhibition, unlike the induction of growth in hypocotyls, requires the presence of the F-box auxin receptors.     

Acclimative changes in root epidermal cell fate in response to Fe and P deficiency: a specific role for auxin?

Summary Root hair formation and the development of transfer cells in the rhizodermis was investigated in various existing auxinrelated mutants ofArabidopsis thaliana and in the tomato mutantdiageotropica. Wild-type Arabidopsis plants showed increased formation of root hairs when the seedlings were cultivated in Fe- or P-free medium. These extranumerary hairs were located in normal positions and in positions normally occupied by nonhair cells, e.g., over periclinal walls of underlying cortical cells. Defects in auxin transport or reduced auxin sensitivity inhibited the formation of root hairs in response to Fe deficiency completely but did only partly affect initiation and elongation of hairs in P-deficient roots. Application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid or the auxin analog 2,4-dichlorophenoxyacetic acid did not rescue the phenotype of the auxin-resistantaxr2 mutant under control and Fe-deficient conditions, indicating that functionalAXR2 product is required for translating the Fe deficiency signal into the formation of extra hairs. The development of extra hairs inaxr2 roots under P-replete conditions was not affected by auxin antagonists, suggesting that this process is independent of auxin signaling. In roots of tomato, growth under Fe-deficient conditions induced the formation of transfer cells in the root epidermis. Transfer cell frequency was enhanced by application of 2,4-dichlorophenoxyacetic acid but was not inhibited by the auxin transport inhibitor N-1-naphthylphthalamic acid. In thediageotropica mutant, which displays reduced sensitivity to auxin, transfer cells appeared to develop in both Fe-sufficient and Fe-deficient roots. Similar to the wild type, no reduction in transfer cell frequency was observed after application of the above auxin transport inhibitor. These data suggest that auxin has no primary function in inducing transfer cell development; the formation of transfer cells, however, appears to be affected by the hormonal balance of the plants.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - TIBA triiodobenzoic acid - NPA N-1-naphthylphthalamic acid - STS silver thiosulfate     

In Plant Protoplasts, the Spontaneous Expression of Defense Reactions and the Responsiveness to Exogenous Elicitors Are under Auxin Control

When auxin was omitted during either the preparation or the culture of tobacco mesophyll protoplasts, as well as during both periods, synthesis of β-glucanase was spontaneously induced. In contrast, when protoplasts were prepared and cultured in the presence of 16 micromolar 1-naphthaleneacetic acid (optimal concentration for protoplast division), the expression of β-glucanase was maintained close to the minimal level observed in tobacco leaves. This inhibitory effect was only promoted by active auxins (1-naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, and 3-indoleacetic acid) but not by inactive auxin analogs. Tobacco protoplasts responded to exogenous elicitors from the cell wall of Phytophthora megasperma glycinea (Pmg) by accumulating β-glucanase in the presence of 16 micromolar 1-naphthaleneacetic acid. At higher auxin concentrations, the elicitor-induced β-glucanase synthesis was inhibited. Naphthaleneacetic acid concentration (3 × 10⁻⁵ molar) required to inhibit by 50% the expression of this defense reaction triggered by a near-optimal elicitor concentration was about 100 times higher than that sufficient to inhibit by 50% the spontaneous expression in nonelicited protoplasts. This is the first demonstration of an auxin-fungal elicitor interaction in the control of a defined defense reaction. The above observations were extended to soybean cell protoplasts. The Pmg elicitor-induced stimulation of the synthesis of pathogenesis related P17 polypeptides and of a 39-kilodalton peptide immunologically related to tobacco β-glucanase was only observed when the spontaneous accumulation of these proteins was inhibited in auxin-treated protoplasts.     

GROWTH AND GRAVITATIONAL RESPONSE IN THE DEVELOPMENT OF LEAF BLADE HYPONASTY

Rapid (4 hr) auxin-induced hyponastic curvature of primary leaves of Phaseolus vulgaris is shown to depend on a positive increase in growth of the lower portion of the blade. The curvature involves laminar growth as well as vein growth and is not due to simple turgor changes. The response is sensitive to gravitational orientation, as inversion and horizontal rotation reduce the auxin-induced curvature. The ethylene-generating compound, 2-chloroethylphosphonic acid, had no hyponastic effect on the leaves when applied to either the upper or lower surface and it inhibited auxin-induced hyponasty. This inhibition was additive to that of inversion. Long-term (24–48 hr) effects of 1 mM auxin depend on the surface of the leaf treated. Application to the upper surface results in epinasty, lower surface application in hyponasty, although the initial response in each case is a hyponastic curvature. A dorsi-ventral auxin transport system and differential auxin sensitivity of upper and lower portions of the leaf blade are postulated to account for these responses.     

Effect of indole-3-acetic acid on cell wall loosening: Changes in mechanical properties and noncellulosic glucose content of Avena coleoptile cell wall

The effect of indole-3-acetic acid on cell wall loosening andchemical modifications of noncellulosic components of the cellwall in Avena coleoptile segments was studied and the followingresults were obtained. (1) Auxin decreased both the minimum stress-relaxation time(T_o) and the noncellulosic glucose content of the cell wall. (2) Decreases were observed in the absence or presence of mannitolsolution at concentrations lower than 0.20 M which osmoticallysuppressed auxin-induced extension, while at concentrationshigher than 0.25 M, there was little auxin effect, indicatingthat it is turgor-dependent. (3) The decrease in T_o of the cell wall and that in the noncellulosicglucose content caused by auxin in the presence of mannitolsolutions of various concentrations paralleled each other (thecorrelation coefficient was 0.897). (4) Both decreases in T_o and glucose content caused by auxinwere inhibited by nojirimycin (5-amino-5-deoxy-D-glucopyranose)in the presence of mannitol. The results suggest that auxin-induced cell wall loosening iscaused by the degradation of noncellulosic rß-glucanin the cell wall. (Received December 24, 1976; )     

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     

The effect of ethylene on auxin-induced growth of excised rice coleoptile segments

Elongation growth induced by exogenous auxin of apical coleoptilesegments of etiolated rice seedlings was promoted by ethylene.In the absence of exogenous auxin, growth promotion was notobserved. The highest promotion by ethylene was obtained at10^–6 M of indole-3-acetic acid, a suboptimal concentrationfor auxin-induced elongation. Level of ethylene which achievedthe effect was less than 1 µl per liter of an incubationatmosphere. ¹Present address: The Ocean Research Institute, University ofTokyo, Nakano, Tokyo, Japan (Received May 27, 1970; )     

Auxin Effects on the Aggregation and Heat Coagulability of Cytoplasmic Proteins and Lipoproteins

The kinetics of induction of heat stability of cytoplasmic proteins and lipoproteins by auxin (2,4,-D) were determined for basal sections of soybean hypocotyl. Maximum heat stabilization occurred after 4 h of tissue incubation with 10^-5M 2,4-D. The effect was less pronounced or absent with longer incubations. Membrane fractions sedimenting between 10,000 and 100,000 g and proteins of the 100,000 g supernatant were most affected. The auxin-induced protein aggregation response varied among experiments. With many tissue lots, the response was small or absent even though the tissue responded to the auxin uniformly by increased growth. The magnitude of response was proportional to the logarithm of auxin concentration but with low 2,4-D the portion of the homogenate protein coagulated by heat was increased and with supraoptimal concentrations it was decreased relative to the control. The smallest auxin-induced change in heat coagulability was observed at the auxin concentration nearest the optimum for growth. No direct correlation was found between the auxin-induced protein and lipo-protein aggregation phenomenon and total protein, chloroform-extractable lipid, residual lipid, growth or tissue deformability. Total sulfhydryl equivalent of the homogenates, however, did correlate with auxin effects on aggregation. This result, plus experiments where homogenates were exposed to oxidizing or reducing conditions, suggests that heat stabilization and associated protein aggregation phenomena are related to conversion of protein sulfhydryl to intramolecular disulfide bonds. No significance is attached to heat stabilization of cytoplasmic proteins as a requisite of auxin-induced growth.     

Ethylene and auxin-induced cell growth in relation to auxin transport and metabolism and ethylene production in the semi-aquatic plant,Regnellidium diphyllum

Cell elongation in the rachis of the semiaquatic fern Regnellidium diphyllum is induced by the addition of ethylene or indoleacetic acid (IAA). Experiments with whole plants or rachis segments have shown that ethylene-induced growth requires the presence of auxin. Ethylene does not cause a modification in either endogenous auxin levels or in the extent of auxin metabolism but auxin transport is reduced. Rates of ethylene production in Regnellidium are not altered by either mechanical excitation or by the addition of auxin. A two-hormone control of cell expansion is proposed in which an initial, auxin-dependent growth event pre-conditions the cells to a further subsequent (or synchronous) ethylene-dependent growth event.Abbreviation IAA indole-3yl-acetic acid