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.     

Stomatal behavior and water relations of waterlogged tomato plants

The effects of waterlogging the soil on leaf water potential, leaf epidermal conductance, transpiration, root conductance to water flow, and petiole epinasty have been examined in the tomato (Lycopersicon esculentum Mill.). Stomatal conductance and transpiration are reduced by 30% to 40% after approximately 24 hours of soil flooding. This is not due to a transient water deficit, as leaf water potential is unchanged, even though root conductance is decreased by the stress. The stomatal response apparently prevents any reduction in leaf water potential. Experiments with varied time of flooding, root excision, and stem girdling provide indirect evidence for an influence of roots in maintaining stomatal opening potential. This root-effect cannot be entirely accounted for by alterations in source-sink relationships. Although 1-aminocyclopropane-1-carboxylic acid, the immediate precursor of ethylene, is transported from the roots to the shoots of waterlogged tomato plants, it has no direct effect on stomatal conductance. Ethylene-induced petiole epinasty develops coincident with partial stomatal closure in waterlogged plants. Leaf epinasty may have beneficial effects on plant water balance by reducing light interception.     

Inhibition of ethylene synthesis in tomato plants subjected to anaerobic root stress

Enhanced ethylene production and leaf epinasty are characteristic responses of tomato (Lycopersicon esculentum Mill.) to waterlogging. It has been proposed (Bradford, Yang 1980 Plant Physiol 65: 322-326) that this results from the synthesis of the immediate precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), in the waterlogged roots, its export in the transpiration stream to the shoot, and its rapid conversion to ethylene. Inhibitors of the ethylene biosynthetic pathway are available for further testing of this ACC transport hypothesis: aminooxyacetic acid (AOA) or aminoethoxyvinylglycine (AVG) block the synthesis of ACC, whereas CO²⁺ prevents its conversion to ethylene. AOA and AVG, supplied in the nutrient solution, were found to inhibit the synthesis and export of ACC from anaerobic roots, whereas Co²⁺ had no effect, as predicted from their respective sites of action. Transport of the inhibitors to the shoot was demonstrated by their ability to block wound ethylene synthesis in excised petioles. All three inhibitors reduced petiolar ethylene production and epinasty in anaerobically stressed tomato plants. With AOA and AVG, this was due to the prevention of ACC import from the roots as well as inhibition of ACC synthesis in the petioles. With Co²⁺, conversion of both root- and petiole-synthesized ACC to ethylene was blocked. Collectively, these data support the hypothesis that the export of ACC from low O₂ roots to the shoot is an important factor in the ethylene physiology of waterlogged tomato plants.     

Role of Ethylene in Induction of Flooding Damage in Sunflower

The possibility that symptoms of flooding damage in plants are primarily caused by an accumulation of ethylene was investigated using pot-grown sunflower (Helianthus annuus) plants. When plants were flooded to the basal pairs of leaves, ethylene in roots and stems below the water line began to increase. This coincided with the start of hypocotyl hypertrophy and new root formation in hypocotyls, which continued for 14-16 days. There were highly significant correlations between ethylene concentration and number of roots and hypocotyl diameter. After approximately 4 days of flooding, ethylene concentrations in stems between nodes for the 1st and 3rd basal pairs of leaves started to increase, coinciding with initiation of chlorophyll breakdown and epinasty of the 2nd basal pairs of leaves. Thus, there were correlations between ethylene concentration and chlorophyll breakdown and epinasty. The lower the leaves, the more chlorophyll breakdown among 1st, 2nd, 3rd, and 4th basal pairs of leaves. The longer the flooding, the more severe the flooding damage; and even when returned to normal condition, plants flooded longer than 3 days were not able to recover from flooding damage. A gas chromatographic study revealed that Ethephon was absorbed by roots and decomposed to ethylene in the plant. Damage symptoms caused by soil application of Ethephon, such as reduced stem height, chlorophyll breakdown, epinasty of the 2nd basal pairs of leaves, and hypocotyl hypertrophy, were almost identical with those caused by soil flooding treatment. Microscopic studies revealed that radially enlarged cells and increased intercellular spaces in the cortex were the major contribution to the increased hypocotyl diameter in both flooded and Ethephon-treated plants. It is concluded that the increase in ethylene concentration in flooded plants is largely, although not exclusively, responsible for flooding damage symptoms.     

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.     

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.     

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 accumulation in waterlogged Rumex plants promotes formation of adventitious roots

Accumulation of the gaseous plant hormone ethylene is very importantfor the induction of several responses of plants to flooding.However, little is known about the role of this gas in the formationof flooding-induced adventitious roots. Formation of adventitiousroots in Rumex species is an adaptation of these plants to floodedsoil conditions. The large air-spaces in these roots enablesdiffusion of gases between shoot and roots. Application of ethylene to non-flooded Rumex plants resultedin the formation of adventitious roots. In R. palustris Sm.shoot elongation and epinasty were also observed. The numberof roots in R. thyrsiflorus Fingerh. was much lower than inR. palustris, which corresponds with the inherent differencein root forming capacity between these two species. Ethyleneconcentrations of 1.5–2µI I_{– 1} induced a maximumnumber of roots in both species. Quantification of ethylene escaping from root systems of Rumexplants that were de-submerged after a 24 h submergence periodshowed that average ethylene concentrations in submerged rootsreached 1.8 and 9.1 µl I_–1 in R. palustris and R.thyrsiflorus, respectively. Inhibition of ethylene productionin R. palustris by L--(2-aminoethoxyvinyl)-glycine (AVG) or-aminobutyric acid (AIB) decreased the number of adventitiousroots induced by flooding, indicating that high ethylene concentrationsmay be a prerequisite for the flooding-induced formation ofadventitious roots in Rumex species. Key words: Adventitious roots, epinasty, ethylene, flooding, Rumex, shoot elongation     

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.     

Cadmium toxicity in tomato (Lycopersicon esculentum) plants grown in hydroponics

The effects of Cd have been investigated in tomato (Lycopersicon esculentum) plants grown in a controlled environment in hydroponics, using Cd concentrations of 10 and 100 μM. Cadmium treatment led to major effects in shoots and roots of tomato. Plant growth was reduced in both Cd treatments, leaves showed chlorosis symptoms when grown at 10 μM Cd and necrotic spots when grown at 100 μM Cd, and root browning was observed in both treatments. An increase in the activity of phosphoenolpyruvate carboxylase, involved in anaplerotic fixation of CO₂ into organic acids, was measured in root extracts of Cd-exposed plants. Also, significant increases in the activities of several enzymes from the Krebs cycle were measured in root extracts of tomato plants grown with Cd. In leaf extracts, significant increases in citrate synthase, isocitrate dehydrogenase and malate dehydrogenase activities were also found at 100 μM Cd, whereas fumarase activity decreased. These data suggest that at low Cd supply (10 μM) tomato plants accumulate Cd in roots and this mechanism may be associated to an increased activity in the PEPC–MDH–CS metabolic pathway involved in citric acid synthesis in roots. Also, at low Cd supply some symptoms associated with a moderate Fe deficiency could be observed, whereas at high Cd supply (100 μM) effects on growth overrule any nutrient interaction caused by excess Cd. Cadmium excess also caused alterations on photosynthetic rates, photosynthetic pigment concentrations and chlorophyll fluorescence, as well as in nutrient homeostasis.     

Effect of aeration on the flood-induced formation of adventitious roots and other changes in sunflower (Helianthus annuus L.)

Summary The effects of flooding, flooding with aeration, and no flooding of the root system on shoot growth was studied in sunflower plants. The responses of shoots appear to be brought about by: (1) The anaerobic condition of the roots which causes stem dwarfing, chlorosis, and petiolar epinasty. (2) The presence of water in excess of field capacity (but not anoxia) around the roots which results in an increase in stem hypertrophy and the formation and growth of adventitious roots.Abbreviations (F, NF, FA) are explained in Material and Methods.     

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.     

Ethanol synthesis and loss from flooded roots of Medicago sativa L. and Lotus corniculatus L.

Abstract Field flooding of established alfalfa (Medicago sativa L.) and birdsfoot trefoil (Lotus corniculatus L.) for up to 12 d resulted in a significant increase in alcohol dehydrogenase activity (ADH) and an increase in the K_m of ADH in both species. Root concentration of ethanol increased throughout the flooding regime in alfalfa roots. No ethanol was detected in any trefoil root samples. Alfalfa plants which had shoots removed 5 d prior to flooding accumulated significantly higher levels of root ethanol and showed flooding injury sooner, indicating a significant effect of shoots on development of flooding injury. Alfalfa and trefoil plants grown in the greenhouse were flooded and ethanol in the transpiration effluent was trapped and measured. Alfalfa transpired measurable quantities of ethanol which peaked just prior to development of shoot injury symptoms. No ethanol was detected in the transpiration effluent from trefoil shoots. Flooded roots of both alfalfa and trefoil excreted ethanol but alfalfa roots synthesized more total ethanol and retained a larger proportion in the roots than did trefoil. While the ethanol accumulation response in alfalfa and trefoil are consistent with the ethanol ‘self-poisoning’ hypothesis of flooding injury, the very small quantities of ethanol found in these roots still raises questions as to its absolute effect in the plant.     

The effect of waterlogging on the growth and ethylene content of Eucalyptus robusta Sm. (Swamp Mahogany)

Summary When waterlogged over a period of 80 days plants of Eucalyptus robusta Sm. showed symptoms of leaf chlorosis, epinasty and premature abscission, reduction of stem elongation, stem hypertrophy and formation of adventitious shoots; chlorophyll content was reduced and soluble protein content of the upper leaves increased. Waterlogging doubled the rate of release of ethylene from roots and stems within 6 days, but had no effect on the ethylene concentration of leaves.     

Ethylene Production by Meloidogyne spp.-Infected Plants

Gall size and rates of ethylene production by various hosts infected with Meloidogyne javanica and by excised tomato root cultures infected with M. javanica or M. hapla were measured. Infection with M. javanica increased the rate of ethylene production in dicotyledonous plants (cabbage, pea, carrot, cucumber, carnation, and tomato), but not in infected monocotyledonous plants (corn, wheat, and onion). Nematode infection induced large galls on roots of dicotyledonous, but not monocotyledonous, plants. Excised tomato roots in culture infected with M. javanica produced ethylene at high rates and formed large galls, whereas roots infected with M. hapla produced ethylene at low rates and induced smaller galls.     

Regulation of epinasty induced by 2,4‐dichlorophenoxyacetic acid in pea and Arabidopsis plants

The herbicide 2,4‐dichlorophenoxyacetic acid (2,4‐D) causes uncontrolled cell division and malformed growth in plants, giving rise to leaf epinasty and stem curvature. In this study, mechanisms involved in the regulation of leaf epinasty induced by 2,4‐D were studied using different chemicals involved in reactive oxygen species (ROS) accumulation (diphenyleniodonium, butylated hydroxyanisole, EDTA, allopurinol), calcium channels (LaCl₃), protein phosphorylation (cantharidin, wortmannin) and ethylene emission/perception (aminoethoxyvinyl glycine, AgNO₃). The effect of these compounds on the epinasty induced by 2,4‐D was analysed in shoots and leaf strips from pea plants. For further insight into the effect of 2,4‐D, studies were also made in Arabidopsis mutants deficient in ROS production (rbohD, rbohF, xdh), ethylene (ein 3‐1, ctr 1‐1, etr 1‐1), abscisic acid (aba 3.1), and jasmonic acid (coi 1.1, jar 1.1, opr 3) pathways. The results suggest that ROS production, mainly ·OH, is essential in the development of epinasty triggered by 2,4‐D. Epinasty was also found to be regulated by Ca²⁺, protein phosphorylation and ethylene, although all these factors act downstream of ROS production. The use of Arabidopsis mutants appears to indicate that abscisic and jasmonic acid are not involved in regulating epinasty, although they could be involved in other symptoms induced by 2,4‐D.     

Epinasty promoted by salinity or ethylene is an indicator of salt-sensitivity in tomatoes

Abstract Six genotypes of tomato (Lycopersicon esculentum Mill.) that differ in their salt-tolerance, were exposed to 200 mol m^?3 NaCl for 4 weeks. Seedlings exhibited a marked decline in shoot dry weight accumulation and increased petiolar epinasty after exposure to salinity stress. Ranking accessions on the basis of their relative growth reduction in response to salinity, provided good agreement with the level of epinasty promoted during the salinity treatment. In the absence of salt-stress, leaf epinasty promoted by exogenous ethylene treatment was found to be a positive indicator of the genotypes incipient salt-sensitivity. Endogenous ethylene levels in untreated plants were negatively correlated with ethephon-induced epinasty. Genotypes with normally high endogenous C₂H₄ levels were less responsive to ethephon treatment and also exhibited greater salt-tolerance than genotypes with low endogenous C₂H₄ levels. These observations are consistent with the suggestion that a main feature of adaptation in the genotypes examined may involve modulation of their cellular sensitivity to C₂H₄. The results indicate that leaf epinasty, whether salt- or ethylene-induced, is a sensitive indicator of salt-sensitivity. Ethylene-induced epinasty may, therefore, provide a simple basis upon which to identify and select salt-tolerant plants.     

Involvement of plant growth substances in the alteration of leaf gas exchange of flooded tomato plants

Ethylene, abscisic acid, and cytokinins were tested for their ability to either induce or prevent the changes which occur in gas exchange characteristics of tomato (Lycopersicon esculentum Mill. cv. Rheinlands Ruhm) leaves during short-term soil flooding. Ethylene, which increases in the shoots of flooded plants, had no effect on stomatal conductance or photosynthetic capacity of drained plants. Abscisic acid, which also accumulates in the shoots of flooded plants, could reproduce the stomatal behavior of flooded plants when sprayed on the leaves of drained plants. However, photosynthetic capacity of drained plants was unaffected by abscisic acid sprays. Cytokinin export from the roots to the shoots declines in flooded plants. Spray applications of benzyladenine increased stomatal conductance in both flooded and drained plants. In addition, the decline in photosynthetic capacity during flooding was largely prevented by supplementary cytokinin applications. The possible involvement of these growth substances in modifying leaf gas exchange during flooding is discussed.     

Effects on plant growth and root-knot nematode infection of an endophytic GFP transformant of the nematophagous fungus Pochonia chlamydosporia

Pochonia chlamydosporia (Pc123) is a fungal parasite of nematode eggs which can colonize endophytically barley and tomato roots. In this paper we use culturing as well as quantitative PCR (qPCR) methods and a stable GFP transformant (Pc123gfp) to analyze the endophytic behavior of the fungus in tomato roots. We found no differences between virulence/root colonization of Pc123 and Pc123gfp on root-knot nematode Meloidogyne javanica eggs and tomato seedlings respectively. Confocal microscopy of Pc123gfp infecting M. javanica eggs revealed details of the process such as penetration hyphae in the egg shell or appressoria and associated post infection hyphae previously unseen. Pc123gfp colonization of tomato roots was low close to the root cap, but increased with the distance to form a patchy hyphal network. Pc123gfp colonized epidermal and cortex tomato root cells and induced plant defenses (papillae). qPCR unlike culturing revealed reduction in fungus root colonization (total and endophytic) with plant development. Pc123gfp was found by qPCR less rhizosphere competent than Pc123. Endophytic colonization by Pc123gfp promoted growth of both roots and shoots of tomato plants vs. uninoculated (control) plants. Tomato roots endophytically colonized by Pc123gfp and inoculated with M. javanica juveniles developed galls and egg masses which were colonized by the fungus. Our results suggest that endophytic colonization of tomato roots by P. chlamydosporia may be relevant for promoting plant growth and perhaps affect managing of root-knot nematode infestations.     

Metabolic adaptation to flooding stress in upland and lowland rice seedlings

Ability of metabolic adaptation in upland and lowland rice (Oryza sativa L.) seedlings to flooding stress was compared. Flooding stress increased alcohol dehydrogenase (ADH) activity and ethanol concentration in shoots and roots of the upland and lowland rice seedlings. The difference in ADH activity and ethanol concentration in shoots between the upland and lowland rice was not apparent. However, both ADH activity and ethanol concentration in roots of the lowland rice were 2-fold greater than those in roots of the upland rice, suggesting that flooding-induction of ethanolic fermentation in lowland rice roots may be significantly greater than that in the upland rice roots. Since flooding often causes the anaerobic conditions in rooting zone than aerial part of plants and ethanolic fermentation is essential to survive in the anaerobic conditions, the ability of metabolic adaptation in lowland rice seedlings to flooding stress may be greater than that in upland rice seedlings.