Auxin and Ethylene Regulation of Petiole Epinasty in Two Developmental Mutants of Tomato, diageotropica and Epinastic

The epinastic growth responses of petioles to auxin and ethylene were quantified in two developmental mutants of tomato (Lycopersicon esculentum Mill.). In the wild type parent line, cultivar VFN8, the epinastic response of excised petiole sections was approximately log-linear between 0.1 and 100 micromolar indole-3-acetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations, with a greater response to 2,4-D at any concentration. When ethylene synthesis was inhibited by aminoethoxyvinylglycine (AVG), epinasty was no longer induced by auxin, but could be restored by the addition of ethylene gas. In the auxin-insensitive mutant, diageotropica (dgt), no epinastic response to IAA was observed at IAA concentrations that effectively induced epinasty in VFN8. In the absence of added IAA, epinastic growth of dgt petioles in 1.3 microliters per liter exogenous ethylene gas was more than double that of VFN8 petioles. IAA had little additional effect in dgt, but promoted epinasty in VFN8. These results confirm that tomato petiole cells respond directly to ethylene and make it unlikely that the differential growth responsible for epinasty results from lateral auxin redistribution. The second mutant, Epinastic (Epi), exhibits constitutively epinasty, cortical swelling, and root branching symptomatic of possible alternation in auxin or ethylene regulation of growth. Only minor quantitative differences were observed between the epinastic responses to auxin and ethylene of VFN8 and Epi. However, in contrast to VFN8, when ethylene synthesis or action was inhibited in Epi, auxin still induced 40 to 50% of the epinastic response observed in the absence of inhibitors. This indicates that the target cells for epinastic growth in Epi are qualitatively different from those of VFN8, having gained the ability to grow differentially in response to auxin alone. The dgt and Epi mutants provide useful systems in which to study the genetic determination of target cell specificity for hormone action.     

Ethylene, seed germination, and epinasty

Ethylene activity in lettuce seed (Lactuca satina) germination and tomato (Lycopersicon esculentum) petiole epinasty has been characterized by using heat to inhibit ethylene synthesis. This procedure enabled a separation of the production of ethylene from the effect of ethylene. Ethylene was required in tomato petioles to produce the epinastic response and auxin was found to be active in producing epinasty through a stimulation of ethylene synthesis with the resulting ethylene being responsible for the epinasty. In the same manner, it was shown that gibberellic acid stimulated ethylene synthesis in lettuce seeds. The ethylene produced then in turn stimulated the seeds to germinate. It was hypothesized that ethylene was the intermediate which caused epinasty or seed germination. Auxin and gibberellin primarily induced their response by stimulating ethylene production.     

Failure of Ethylene to Change the Distribution of Indoleacetic Acid in the Petiole of Coleus blumei X frederici during Epinasty

The effect of ethylene on the distribution of applied indoleacetic acid in the petiole of Coleus blumei Benth. X C. frederici G. Taylor has been investigated during the development of epinastic curvature. Using intact plants, ¹⁴C-IAA was applied to the distal region of the leaf lamina and the accumulation of label in the abaxial and adaxial halves of 5 mm petiole sections was determined after 1.5, 3, and 6 hours. Over this period the label was transported out of the lamina into the petiole at a rate of at least 66 mm hr⁻¹. Of the total amount of label in the petiole sections, 24 to 30% was located in the adaxial half and this distribution was not altered significantly by exposing plants to an atmosphere containing 50 μl/l ethylene. Thus when epinastic curvature is induced by ethylene there is no associated increase in the IAA content of the expanding adaxial half. The role of endogenous IAA in petiole epinasty was studied by restricting its movement with DPX 1840 (3,3a-dihydro-2-[p-methoxyphenyl]-8H-pyrozolo{5,1-a}isoindol-8-one). The leaf petioles still showed an initial epinastic response to ethylene. It is concluded that ethylene-induced epinasty is not dependent upon either any change in the transport of IAA or its redistribution within the petiole.     

Brassinosteroid-induced epinasty in tomato plants

The effects of root treatments of brassinosteroid (BR) on the growth and development of hydroponically grown tomato plants (Lycopersicon esculentum Mill cv Heinz 1350) were evaluated. There was a dramatic increase in petiole bending when the plants were treated with 0.5 to 1.0 micromolar BR. The leaf angle of the treated plants was almost three times that of untreated controls. BR-induced epinasty appeared to be due to stimulation of ethylene production. Excised petioles from BR-treated plants produced more than twice as much ethylene as did untreated controls. As ethylene production increased, the degree of petiole bending also increased, and inhibition of ethylene production by AOA or CoCl₂ also inhibited epinasty. BR-treated plants had increased levels of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) in the leaf tissue. ACC appeared to accumulate primarily in the petioles with the greatest amount of ACC accumulating in the youngest petioles. Time course evaluations revealed that BR treatment stimulated ACC production. As ACC accumulated, ethylene increased, resulting in epinasty. Little or no ACC was found in the xylem sap, indicating that there was a signal transported from the roots which stimulated ACC synthesis in the leaf tissue.     

Gravity can induce epinastic bending of isolated leaves

Abstract Isolated leaves of Plectranthus fruticosus were grown in cubic plastic cuvettes, and were supplied via their cut petioles with nutrient solution and indole-3-acetic acid (10^?6m ). Holes bored in the cuvette walls allowed the petioles to be oriented at approximately 60°, 90° or 120° to the vertical. Growth of the leaves initially oriented at angles of 60° and 90°, which simulated the situation in the intact plant, did not result in epinastic bending of the petiole. Inversion of the leaves (adaxial surface of the petiole downwards) and orientation of the adaxial/abaxial surfaces of the horizontal petiole parallel to the gravity vector, however, yielded strong epinastic bending of the petioles. In the latter case, this bending was not in the direction of the gravity vector (evidence for point (iii), below). Furthermore, epinastic bending occurred, when the isolated leaves were rotated on a clinostat (petioles parallel to the rotation axis or inclined to the rotation axis at an angle of 30°; 3 r.p.m.). Since a possible influence of the shoot was excluded, it is concluded that (i) perception and response are restricted to the leaf, (ii) gravity alone is sufficient to induce epinasty, (iii) a gravitropic component of the response can probably be excluded. The clinostat induced epinasty may not have been caused by nullifying the effect of gravity but due to continuous gravistimulation of the leaf.     

Prevention of salt-induced epinasty by α-aminooxyacetic acid and cobalt

Ethylene production in leaf petiole and laminae tissues was stimulated in tomato (Lycopersicon esculentum Mill. cv. UCT5) plants exposed to salinity-stress. At the highest salinity level (250 mM NaCl), rates of ethylene production more than doubled over those observed in non-stressed plants. Correspondingly, petiolar epinasty increased with increasing levels of stress impositions. Both responses were suppressed when either 1 mM -aminooxyacetic acid (AOA), or 100 M Co²⁺ was simultaneously applied. Co²⁺, but not AOA, had a pronounced effect on ethylene production resulting from the application of a saturating dose (2 mM) of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene. This result suggests that ethylene production is dependent upon the activity of ethylene forming enzyme (EFE). The magnitude of ethylene stimulation in leaf petioles was related to the salinity level imposed and to the induction of petiole epinasty. In the absence of stress impositions, epinastic responsiveness to ethylene or its precursor, ACC, might provide a simple, indirect criteria to adjudge salt-sensitivity among plants.Research supported by AID contract II, NEB-1070-A-00-2074-00.     

Effects of root anaerobiosis on ethylene production, epinasty, and growth of tomato plants

Experiments were performed to determine the source(s) of ethylene-causing epinasty in flooded tomato plants (Lycopersicon esculentum Mill.). Simultaneous measurements were made of ethylene synthesized by the roots and shoots of tomato plants exposed to either aerobic or anaerobic atmospheres in the root zone. When the root zone was made anaerobic by a flowing stream of N₂ gas, petiole epinasty and accelerated ethylene synthesis by the shoots were observed. In soil-grown plants, ethylene synthesis by the root-soil complex increased under anaerobic conditions; but when grown in inert media under the same conditions, ethylene synthesis by roots remained constant or declined during the period of rapid epinastic growth by the petioles. Other characteristic symptoms of flooding, e.g. reduced growth and chlorosis, were also observed in plants with anaerobic roots. Pretreatment of plants with AgNO₃, an inhibitor of ethylene action, completely prevented epinasty, demonstrating that ethylene is the agent responsible for waterlogging symptoms. These results indicate that deprivation of O₂ to the roots is the primary effect of soil flooding, and that this is sufficient to cause increased ethylene synthesis in the shoot. The basis of the observed root-shoot communication is unknown, but root-synthesized hormones or specific ethylene-promoting factors may be involved.     

Xylem Transport of 1-Aminocyclopropane-1-carboxylic Acid, an Ethylene Precursor, in Waterlogged Tomato Plants

Waterlogging is known to cause an increase in ethylene synthesis in the shoot which results in petiole epinasty. Evidence has suggested that a signal is synthesized in the anaerobic roots and transported to the shoot where it stimulates ethylene synthesis. Experimental data are presented showing that 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, serves as the signal. Xylem sap was collected from detopped tomato plants (Lycopersicon esculentum Mill. cv. VFN8). ACC in the sap was quantitated by a sensitive and specific assay, and its tentative chemical identity verified by paper chromatography. ACC levels in both roots and xylem sap increased markedly in response to waterlogging or root anaerobiosis. The appearance of ACC in the xylem sap of flooded plants preceded both the increase in ethylene production and epinastic growth, which were closely correlated. Plants flooded and then drained showed a rapid, simultaneous drop in ACC flux and ethylene synthesis rate. ACC supplied through the cut stem of tomato shoots at concentrations comparable to those found in xylem sap caused epinasty and increased ethylene production. These data indicate that ACC is synthesized in the anaerobic root and transported to the shoot where it is readily converted to ethylene.     

Ethylene as a Cause of Transient Petiole Epinasty in Helianthus annuus during Clinostat Experiments

Petiole curvature and elongation growth in Helianthus annuus L. have been recorded for plants rotating with their stems parallel to the horizontal axis of a clinostat at 8 revolutions per hour over 72 hours. When rotation was continuous, dorso-convex curvature (epinasty) developed in the first 12 hours and was followed by recovery (straightening) in the next 36 hours. Thereafter the petioles remained straight. These changes in shape are due to brief consecutive increases in the elongation growth of the upper and lower halves of the petiole. Plants exposed to 10 μl per liter ethylene after 200 hours on the clinostat, developed similar petiole epinasty, followed by straightening when the exposure to ethylene ceased. Interrupting rotation of the plant for 1 hour in 4, did not change the petiole response, whereas the alternation of 4 hour stationary and rotation periods, delayed the straightening process. The axillary angle between the stem and petiole increased from about 40° to 63° during either continuous rotation or rotation with 1 or 4 hour stationary periods. When detached leaves were inverted, the rate of ethylene release approximately doubled after 4 hours and continued to increase thereafter. The results indicate that the development of transient petiole epinasty on the clinostat, is due to ethylene production caused primarily by the disorientation of the plant, rather than to the rotation process.     

Is the Diageotropic Tomato Ethylene Deficient?

The production of ethylene by a mutant form of tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig) with diageotropic shoot growth was compared with that from non-mutant, upright plants. No difference in the rate of production by segments of petiole or stem apex was observed. The amounts of ethylene produced by excised segments of petiole from diageotropic and upright plants in response to wounding were also comparable. When the roots of either kind of plant were exposed to anaerobic conditions, the production of ethylene increased in the petioles; in ordinary plants this was associated with epinastic curvature, while in diageotropic plants the direction of shoot extension became reorientated from the horizontal to a more upright position. Exogenous ethylene gas had similar effects. These results support the view that the mutant has a modified response mechanism to gravity and to ethylene rather than an abnormally slow rate of ethylene production in the shoot. Since applying inhibitors of ethylene action to non-mutant, upright plants did not induce diageotropism, endogenous ethylene seems unlikely to play a significant role in maintaining their upright orientation. The roots of both kinds of plant produced large amounts of ethylene, although the rate for diageotropic roots was about 37% less than that of roots from normal plants. Application of indol-3-ylacetic acid increased the production of ethylene by all roots but those from mutant plants were less responsive.     

Effects of Mecoprop (an Auxin Analogue) on Ethylene Evolution and Epinasty in Two Biotypes of Stellaria media

Petiolar epinasty and the production of ethylene (ethene) werestudied in chickweed biotypes, Stellaria media, treated withthe herbicide and auxin analogue (RS)-2-(4-chloro-o-tolyloxy)propionicacid, potassium salt, common name mecoprop. This compound causedsevere epinasty and stimulated the production of ethylene fromshoot explants. However, when intact plants were treated withethylene, the leaves became only slightly epinastic. The ethyleneprecursor, 1-aminocyclopropane-I-carboxylic acid (ACC), at concentrationswhich stimulated the release of ethylene, was equally ineffectivein causing epinasty. Furthermore, 2, 5-norbornadiene, a specific,competitive inhibitor of ethylene action, only partly alleviatedmecoprop-induced epinasty. The responses observed in chickweedwere compared with those produced in tomato plants. ACC inducedepinasty in tomato within 2 h and these symptoms were completelyinhibited by norbornadiene. However, as in chickweed, the inhibitorgave only partial reversal of mecoprop-induced epinasty, implyingthat the epinastic response caused by the herbicide was notattributable to ethylene alone. We therefore suggest that mecoprop-inducedepinasty is a result of the combined ethylene-stimulating andgrowth-promoting properties of the herbicide. Mecoprop-stimulated ethylene evolution was initially significantlygreater in a herbicide-resistant, compared with a more susceptiblebiotype of chickweed. The significance of this finding is discussedin relation to the mechanism of mecoprop resistance in chickweed. Epinasty, ethylene, (RS)-2-(4-chloro-o-tolyloxy)propionic acid, mecoprop, herbicide resistance, chickweed, Stellaria media L., tomato, Lycopersicon esculentum L.     

Increased Ethylene Production during Clinostat Experiments May Cause Leaf Epinasty

Ethylene production from tomato (Lycopersicum esculentum L. cv. Rutgers) plants based on a clinostat doubled during the first 2 hours of rotation. Carbon dioxide blocked the appearance of leaf epinasty normally associated with plants rotated on a clinostat. These results support the idea that epinasty of clinostated plants was due to increased ethylene production and not to the cancellation of the gravitational pull on auxin transport in the petiole.     

Characterization of an Ethylene Overproducing Mutant of Tomato (Lycopersicon esculentum Mill. Cultivar VFN8)

Ethylene production rates and tissue ethylene concentrations were determined for the single-gene, Epinastic (Epi) tomato (Lycopersicon esculentum Mill.) mutant, and its parent, cv VFN8. The Epi phenotype was characterized by severe leaf epinasty, thickened stems and petioles, and a compact growth habit. In 4-day-old seedlings, ethylene production was significantly higher in Epi than in VFN8. Ethylene production rates also were higher for excised root, hypocotyl, cotyledon, and shoot tissue of 14-day-old Epi seedlings as compared with VFN8. The greatest difference in the ethylene production rate was observed in excised Epi shoot tissue, which was more than 2.5 times higher than in VFN8. Tissue ethylene concentrations of 19−, 25−, and 31-day-old Epi plants were 8, 172, and 307% higher than for VFN8, corresponding to increasing expression of the Epi phenotypic characteristics with age. The highest ethylene concentrations occurred in the shoot apex of both genotypes. Higher ethylene concentrations in Epi resulted from greater 1-aminocyclopropane-1-carboxylic acid content rather than increased ethylene-forming enzyme activity. The elevated ethylene levels in Epi did not result from increased auxin sensitivity. The sensitivity of root growth to inhibition by ethylene did not differ between VFN8 and Epi. Although elevated levels of ethylene in Epi plants apparently exacerbate its epinastic growth characteristics, other evidence indicates that this may not be the fundamental lesion. This mutant may provide a unique system for investigating the regulation of ethylene biosynthesis and the role of target cell types in plant development.     

Ethylene-induced opposite redistributions of calcium and auxin are essential components in the development of tomato petiolar epinastic curvature.

Calcium has been suggested as an important mediator of gravity signaling transduction within the root cap statocyte. In a horizontally-placed root, it is redistributed in the direction of the gravity vector (i.e. it moves downward) and its redistribution is closely correlated with auxin downward movement. However, the involvement of calcium in the regulation of ethylene-induced epinasty and auxin movement is not known. In this report, we examined the involvement of calcium in lateral auxin transport during ethylene-induced epinasty in an effort to understand the relationship among calcium, auxin, and ethylene. Ethylene-induced epinasty was further stimulated by exogenously applied Ca2+, the calcium effect being the strongest among divalent cations tested. Pretreatment with NPA, an auxin transport inhibitor, negated the promotive effect of calcium ions on the petiolar epinasty. Ethylene caused redistribution/differential accumulation of 45Ca2+ toward the morphologically lower (abaxial) side of the leaf petioles, an effect opposite to that of 14C-IAA redistribution. Verapamil, a Ca2+ channel blocker, inhibited ethylene-induced epinasty, as well as the redistribution of 14C-IAA and 45Ca2+. When the petiole was inverted in the presence or absence of ethylene, the direction of 45Ca2+ differential accumulation was still toward the morphologically abaxial side of the petiole during epinastic movement regardless of gravitational direction. These results suggest that gravity-insensitive, ethylene-induced Ca2+ redistribution and accumulation toward the abaxial side are closely coupled to the adaxial auxin redistribution/accumulation and, in turn, to the petiolar epinasty.     

Inhibition of brassinosteroid-induced epinasty in tomato plants by aminooxyacetic acid and Co2+

The inhibitory effects of aminooxyacetic acid (AOA) and cobalt chloride (CoCl₂) on brassinosteroid (BR)-induced epinasty in tomato plants ( Lycopersicon esculentum Mill. cv. Heinz 1350) are evaluated. CoCl₂ dramatically decreases petiole bending and ethylene production as the concentration increases from 50 to 200 μ M. The content of 1-aminocyclopropane-1-carboxylic acid (ACC) in the petiole, instead of accumulating, is reduced and does not change over the concentration range tested. Inhibition of BR-induced epinasty by AOA results from inhibition of ACC synthesis. There are dramatic reductions in petiole bending, ethylene and ACC production as the concentration of AOA is increased from 50 to 200 μ M. Maximum inhibition occurs when the plants are pretreated with the inhibitors. The degree of inhibition increases as the length of pretreatment increases from 1 to 4 h. The response of BR-treated plants to AOA and CoCl₂ is similar to the effect of auxin, indicating the integral relationship between BR and auxin.     

Effect of Waterlogged Soil Conditions on the Production of Ethylene and on Water Relationships in Tomato Plants

The roots of tomato plants (Lycopersicon esculentum Mill., cv.Moneymaker) were exposed to low concentrations of oxygen bywaterlogging the soil or by growing the plants in nutrient solutionflushed with nitrogen gas. After 24 h, the rate of ethyleneproduction by the petioles, main stem, and shoot apex was increasedby 4–6-fold and the petioles developed epinastic curvatures.Removing the roots did not reproduce these responses. The amountsof ethylene produced by shoot tissues in response to physicalwounding was greatly increased by waterlogging the soil. The production of ethylene by roots was suppressed by the absenceof oxygen. When the roots were transferred back to an aerobicenvironment ethylene production quickly exceeded that observedin roots maintained continuously in aerobic conditions. The enhanced rate of ethylene production in the shoots occurredin the absence of increased water stress as measured with aleaf pressure chamber; leaf water potentials were increasedrather than decreased by waterlogging for 30 h or more. Thiswas associated with stomatal closure and reduced transpiration.Resistance to water flow through the plant increased as transpirationdecreased in response to waterlogging. However, at similar ratesof transpiration, resistance was normally lower in waterloggedplants than in controls.     

Prevention of auxin-induced epinasty by α-aminooxyacetic acid

Elhylene production and epinastic growth of leaf petioles of tomato (Lycopersicon esculentum Mill. cv. Moneymaker) plants sprayed with 0.1 mM naphthyl-1-acetic acid were suppressed when 1 mMα-aminooxyacetic acid (AOA) was simultaneously sprayed on the plants. AOA had no effect on ethylene evolution and epinastic growth resulting from the application of 5 mM 1-aminocyclopropane-1-carboxylic acid, the immediate precursor of ethylene.     

Interpreting Plant Responses to Clinostating: I. MECHANICAL STRESSES AND ETHYLENE

The severe epinasty and other symptoms developed by clinostated leafy plants could be responses to gravity compensation and/or the mechanical stresses of leaf flopping. Epinasty in cocklebur (Xanthium strumarium L.), tomato (Lycopersicon esculentum Mill.), and castor bean (Ricinus communis L.) is delayed by inhibitors of ethylene synthesis and action (aminoethoxyvinylglycine and Ag⁺), confirming the role of ethylene in clinostat epinasty. To test the possibility that clinostat mechanical stresses (leaf flopping) cause ethylene production and, thus, epinasty, vertical plants were stressed with constant, gentle, horizontal, or vertical shaking or with a quick, back-and-forth rotation (twisting). Clinostat leaf flopping was closely approximated but with a minimum of gravity compensation, by turning plants so their stems were horizontal, rotating them quickly about the stem axis, and then returning them to the vertical, repeating the treatment every four minutes (clinostat rotation time). None of these mechanical stresses produced significant epinasties, but vigorous hand-shaking (120 seconds per day) generated minor epinasties, as did Ag⁺ applied daily (concentrations high enough to cause leaf browning). Plants gently inverted every 20 minutes developed epinasty at about the same rate and to about the same extent as clinostated plants, but plants inverted every 20 minutes and immediately returned to the upright position did not become epinastic. It is concluded that clinostat epinasty is probably caused by disturbances in the gravity perception mechanism, rather than by leaf flopping.     

Inhibition of the indole-3-acetic acid-induced epinastic curvature in tobacco leaf strips by 2,4-dichlorophenoxyacetic acid

It has been reported that auxin induces an epinastic growth response in plant leaf tissues. Leaf strips of tobacco (Nicotiana tabacum L. 'Bright Yellow 2') were used to study the effects of indole-3-acetic acid (IAA), the principal form of auxin in higher plants, and a synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-D), on epinastic leaf curvature. Incubation of leaf strips with 10 micro M IAA resulted in a marked epinastic curvature response. Unexpectedly, 2,4-D showed only a weak IAA-like activity in inducing epinasty. Interestingly, the presence of 2,4-D resulted in inhibition of the IAA-dependent epinastic curvature. In vivo Lineweaver-Burk kinetic analysis clearly indicated that the interaction between IAA and 2,4-D reported here is not a result of competitive inhibition. Using kinetic analysis, it was not possible to determine whether the mode of interaction between IAA and 2,4-D was non-competitive or uncompetitive. 2,4-D inhibits the IAA-dependent epinasty via complex and as yet unidentified mechanisms.     

THE INTERACTION OF AUXIN AND ETHYLENE IN THE MAINTENANCE OF LEAF BLADE FORM IN PHASEOLUS VULGARIS L. VAR. PINTO

Auxin treatment results in hyponastic curvature of the primary leaves of Phaseolus vulgaris L. var pinto. Ethylene production by hyponastic leaves is detected within 1 hr after treatment with IAA in concentrations at or above 1 μm. The amount of ethylene detected is proportional to the concentration of auxin applied. Untreated control leaves and leaves treated with 2,3,5-tri-iodobenzoic acid or gibberellic acid did not produce ethylene detectable by our equipment. The hyponastic curvature induced by auxin treatment can be inhibited by exogenous application of ethylene or ethylene-generating compounds, and these treatments produce epinasty in auxin-treated leaves. Treatment with inhibitors of ethylene synthesis or action, such as aminoethoxy-vinylglycine, carbon dioxide, or heat treatment, prolong hyponasty. The planar form, therefore, appears to be affected by both hyponastic auxin effect and an epinastic ethylene effect.