Contrasting interactions between ethylene and abscisic acid in Rumex species differing in submergence tolerance

Complete submergence of flooding-tolerant Rumex palustris plants strongly stimulates petiole elongation. This escape response is initiated by the accumulation of ethylene inside the submerged tissue. In contrast, petioles of flooding-intolerant Rumex acetosa do not increase their elongation rate under water even though ethylene also accumulates when they are submerged. Abscisic acid (ABA) was found to be a negative regulator of enhanced petiole growth in both species. In R. palustris, accumulated ethylene stimulated elongation by inhibiting biosynthesis of ABA via a reduction of RpNCED expression and enhancing degradation of ABA to phaseic acid. Externally applied ABA inhibited petiole elongation and prevented the upregulation of gibberellin A(1) normally found in submerged R. palustris. In R. acetosa submergence did not stimulate petiole elongation nor did it depress levels of ABA. However, if ABA concentrations in R. acetosa were first artificially reduced, submergence (but not ethylene) was then able to enhance petiole elongation strongly. This result suggests that in Rumex a decrease in ABA is a prerequisite for ethylene and other stimuli to promote elongation.     

Ethylene Sensitivity and Response Sensor Expression in Petioles of Rumex Species at Low O2 and High CO2 Concentrations

Rumex palustris, a flooding-tolerant plant, elongates its petioles in response to complete submergence. This response can be partly mimicked by enhanced ethylene levels and low O2 concentrations. High levels of CO2 do not markedly affect petiole elongation in R. palustris. Experiments with ethylene synthesis and action inhibitors demonstrate that treatment with low O2 concentrations enhances petiole extension by shifting sensitivity to ethylene without changing the rate of ethylene production. The expression level of the R. palustris gene coding for the putative ethylene receptor (RP-ERS1) is up-regulated by 3% O2 and increases after 20 min of exposure to a low concentration of O2, thus preceding the first significant increase in elongation observable after 40 to 50 min. In the flooding-sensitive species Rumex acetosa, submergence results in a different response pattern: petiole growth of the submerged plants is the same as for control plants. Exposure of R. acetosa to enhanced ethylene levels strongly inhibits petiole growth. This inhibitory effect of ethylene on R. acetosa can be reduced by both low levels of O2 and/or high concentrations of CO2.     

Growth responses of Rumex species in relation to submergence and ethylene

Abstract. Submergence stimulates growth of the petioles of Rumex palustris and Rumex crispus under field, greenhouse and laboratory conditions. Growth of Rumex acetosa petioles was hardly influenced by submergence. These growth responses under flooded conditions can be partially mimicked by exposing non-submerged Rumex plants to ethylene-air mixtures. Submergence of intact plants in a solution of AgNO₃ inhibited the elongation of all petioles of R. palustris and the youngest petiole of R. crispus and stimulated growth of the youngest petiole of R. acetosa , The ethylene-air mixture experiments, the effect of AgNO₃ and observed increase of the endogenous ethylene concentration during submergence suggest that ethylene plays a regulatory role in the growth responses of these Rumex species under submerged conditions. The three Rumex species showed a gradient in elongation responses to submergence, which correlates with the field distribution of the three species in a flooding gradient.     

Submergence-Induced Ethylene Synthesis,Entrapment, and Growth in Two Plant Species with Contrasting Flooding Resistances

Submergence-induced ethylene synthesis and entrapment were studied in two contrasting Rumex species, one flood-resistant (Rumex palustris) and the other flood-sensitive (Rumex acetosa). The application of a photoacoustic method to determine internal ethylene concentrations in submerged plants is discussed. A comparison with an older technique (vacuum extraction) is described. For the first time ethylene production before, during, and after submergence and the endogenous concentration during submergence were continuously measured on a single intact plant without physical perturbation. Both Rumex species were characterized by enhanced ethylene concentrations in the shoot after 24 h of submergence. This was not related to enhanced synthesis but to continued production and physical entrapment. In R. palustris, high endogenous ethylene levels correlated with enhanced petiole and lamina elongation. No dramatic change in leaf growth rate was observed in submerged R. acetosa shoots. After desubmergence both species showed an increase in ethylene production, the response being more pronounced in R. palustris. This increase was linked to the enhanced postsubmergence growth rate of leaves of R. palustris. Due to the very rapid escape of ethylene out of desubmerged plants to the atmosphere (90% disappeared within 1 min), substantial underestimation of internal ethylene concentrations can be expected using more conventional vacuum extraction techniques.     

The contrasting role of auxin in submergence-induced petiole elongation in two species from frequently flooded wetlands

The involvement of auxin in the submergence-induced petiole elongation has been investigated in Rumex palustris and Ranunculus sceleratus. Both wetland species are capable of enhanced petiole elongation upon submergence or treatment with exogenous ethylene (5μl l⁻¹). Treatment of intact Rumex palustris plants with 1-naphthalene acetic acid (NAA) at 10⁻⁴ M enhanced petiole elongation, while treatment with N -1-naphthylphthalamic acid (NPA) had no effect on petiole elongation. The elongation response after NAA or NPA treatment was comparable for plants in both submerged and drained conditions. Pre-ageing of detached petioles of Rumex palustris for 3 h in light or in dark conditions had no effect on the submergence-induced elongation. In comparison to intact plants, detached petioles of Rumex palustris , with or without lamina, did not show significant differences in responsiveness to IAA between drained or submerged conditions. This was in contrast to Ranunculus sceleratus where submergence caused a clear increase in responsiveness towards IAA. Removal of the lamina, the putative source of auxin, or treatment with NPA did not hinder the submergence-induced elongation of detached Rumex palustris petioles, but severely inhibited elongation of detached Ranunculus sceleratus petioles. This inhibition could be restored by application of NAA, suggesting the specific involvement of auxin in the submergence response of Ranunculus sceleratus. It is concluded that, in contrast to Ranunculus sceleratus , auxin is probably not involved in the submergence-induced petiole elongation of Rumex palustris.     

De-submergence-induced ethylene production in Rumex palustris: regulation and ecophysiological significance

Rumex palustris responds to total submergence by increasing the elongation rate of young petioles. This favours survival by shortening the duration of submergence. Underwater elongation is stimulated by ethylene entrapped within the plant by surrounding water. However, abnormally fast extension rates were found to be maintained even when leaf tips emerged above the floodwater. This fast post-submergence growth was linked to a promotion of ethylene production that is presumed to compensate for losses brought about by ventilation. Three sources of ACC contributed to post-submergence ethylene production in R. palustris: (i) ACC that had accumulated in the roots during submergence and was transported in xylem sap to the shoot when stomata re-opened and transpiration resumed, (ii) ACC that had accumulated in the shoot during the preceding period of submergence and (iii) ACC produced de novo in the shoot following de-submergence. This new production of ethylene was associated with increased expression of an ACC synthase gene (RP-ACS1) and an ACC oxidase gene (RP-ACO1), increased ACC synthase activity and a doubling of ACC oxidase activity, measured in vitro. Out of seven species of Rumex examined, a de-submergence upsurge in ethylene production was seen only in shoots of those that had the ability to elongate fast when submerged.     

Ethylene Biosynthesis and Accumulation under Drained and Submerged Conditions (A Comparative Study of Two Rumex Species)

A model is presented of the regulation of ethylene biosynthesis in relation to submergence and flooding resistance. It is based on time-course measurements of ethylene production, ethylene accumulation, and concentrations of free and conjugated 1-aminocyclo-propane-1-carboxylic acid (ACC) in submerged and drained flooding-resistant Rumex palustris Sm. and flooding-sensitive Rumex acetosella L. plants. From these data, in vivo reaction rates of the final steps in the ethylene biosynthetic pathway were calculated. According to our model, submergence stimulates ACC formation and inhibits conversion of ACC to ethylene in both Rumex species, and as a result, ACC accumulates. This may explain the stimulated ACC conjugation observed in submerged plants. Although submergence inhibited ethylene production, physical entrapment increased endogenous ethylene concentrations in both flooding-resistant R. palustris and flooding-sensitive R. acetosella plants. However, R. palustris plants controlled their internal ethylene levels in the long term by a negative regulation of ACC synthase induced by ethylene. In flooding-sensitive R. acetosella plants, absence of negative regulation increased internal ethylene levels to more than 20 [mu]L L-1 after 6 d of submergence. This may accelerate the process of senescence and contribute to their low level of flooding resistance.     

The stimulating effects of ethylene and auxin on petiole elongation and on hyponastic curvature are independent processes in submerged Rumex palustris

The flooding-tolerant plant species Rumex palustris (Sm.) responds to complete submergence with stimulation of petiole elongation mediated by the gaseous hormone ethylene. We examined the involvement of auxin in petiole elongation. The manipulation of petiolar auxin levels by removing the leaf blade, or by addition of synthetic auxins or auxin transport inhibitors, led to the finding that auxin plays an important role in submergence-induced petiole elongation in R. palustris. A detailed kinetic analysis revealed a transient effect of removing the auxin source (leaf blade), explaining why earlier studies in which less frequent measurements were taken failed to identify any role for auxin in petiole elongation. We previously showed that the onset of stimulated petiole elongation depends on a more upright petiole angle being reached by means of hyponastic (upward) curvature, a differential growth process that is also regulated by ethylene and auxin. This raised the possibility that both ethylene and auxin stimulate elongation only indirectly by influencing hyponastic growth. We show here that the action of ethylene and auxin in promoting petiole elongation in submerged R. palustris is independent of the promoting effect that these hormones also exert on the hyponastic curvature of the same petiole.     

Tracing and interpreting fine-scale human impact in northern Fennoscandia with the aid of modern pollen analogues

A set of moss samples (n=58) was collected and analysed to obtain modern pollen analogues for both natural and human induced vegetation types in northern Fennoscandia. Vegetation types with settlement, trampling and grazing were selected from the different latitudinal forest zones (birch, pine, and mixed forest). The moss species varied between samples but the size of the sample was kept constant. Numerical analyses such as Redundancy Analysis (RDA) indicate that in northern areas human presence is mainly visible as increased values of Gramineae, Rumex acetosa/acetosella, R. obtusifolius, Solidago-type and Achillea-type pollen. Partial RDA further reveals that settlement is strongly correlated with high values of Rumex acetosa/acetosella, whereas trampled sites are significantly characterised by Gramineae and, to a smaller extent, Cyperaceae pollen. It is therefore possible to distinguish the impact of different types of human activities on vegetation on the basis of the pollen spectra. Later, these data will be used to interpret the presence, nature, and duration of human impact from fossil records from the same area. Received June 6, 2001 / Accepted December 12, 2001     

The roles of ethylene, auxin, abscisic acid, and gibberellin in the hyponastic growth of submerged Rumex palustris petioles

Rumex palustris responds to complete submergence with upward movement of the younger petioles. This so-called hyponastic response, in combination with stimulated petiole elongation, brings the leaf blade above the water surface and restores contact with the atmosphere. We made a detailed study of this differential growth process, encompassing the complete range of the known signal transduction pathway: from the cellular localization of differential growth, to the hormonal regulation, and the possible involvement of a cell wall loosening protein (expansin) as a downstream target. We show that hyponastic growth is caused by differential cell elongation across the petiole base, with cells on the abaxial (lower) surface elongating faster than cells on the adaxial (upper) surface. Pharmacological studies and endogenous hormone measurements revealed that ethylene, auxin, abscisic acid (ABA), and gibberellin regulate different and sometimes overlapping stages of hyponastic growth. Initiation of hyponastic growth and (maintenance of) the maximum petiole angle are regulated by ethylene, ABA, and auxin, whereas the speed of the response is influenced by ethylene, ABA, and gibberellin. We found that a submergence-induced differential redistribution of endogenous indole-3-acetic acid in the petiole base could play a role in maintenance of the response, but not in the onset of hyponastic growth. Since submergence does not induce a differential expression of expansins across the petiole base, it is unlikely that this cell wall loosening protein is the downstream target for the hormones that regulate the differential cell elongation leading to submergence-induced hyponastic growth in R. palustris.     

Resistance to complete submergence in Rumex species with different life histories: the influence of plant size and light

Resistance to complete submergence was tested in three Rumex species that occur in the Dutch river forelands. The species differ in both habitat and life history characteristics. The annual or biennial R. maritimus and the biennial or short lived perennial R. palustris grow on frequently flooded mud flats of low elevation, while the perennial R. thyrsiflorus can be found on dykes and river dunes that are seldom flooded. The flooding characteristics of the habitats of the three species were determined. These data were used to design experiments to determine the survival and biomass development of the three species during submergence and the influence of plant size and light level on these parameters. It was shown in all three species that plants submerged during daytime were much more resistant to flooding than those submerged at night. This is most probably due to the generation of oxygen or carbohydrates by underwater photosynthesis. Mature plants of the three species showed higher survival after submergence than juvenile plants, which might be caused by higher carbohydrate levels in the taproots of mature plants. In addition, the three species clearly differed in survival and biomass development during submergence. Rumex thyrsiflorus , the species least subjected to flooding, is least tolerant to complete submergence. Rumex maritimus , which can avoid the floods by having a short life cycle, is less tolerant to submergence than R. palustris , which has to survive the floods as a vegetative plant. It was noted that some plants that survived the flooding period itself, still died in the following period of drained conditions, possibly due to post-anoxic injury.     

Nitrification in the rhizosphere of a flooding-resistant and a flooding-non-resistant Rumex species under drained and waterlogged conditions

Abstract The effects of the flooding-resistant plant species Rumex palustris and the non-flooding-resistant plant species Rumex acetosa on nitrification were compared. The plants were grown under drained and waterlogged conditions on a mixture of calcareous riversand and sieved grassland soil with a high potential nitrifying activity. In the shoots of R. acetosa , but not in those of R. palustris , the ratio between the amounts of accumulated carboxylates and organic nitrogen, ((CA-A)/N_org.), appeared to be a useful indicator of ammonium or nitrate consumption by tghe plant. In both plant species, the inorganic nitrogen source had no observed effect on the (C-A)/N_org. ratio in the roots.
The growth of R. acetosa , but not that of R. palustris was inhibited by waterlogging of the soil. Both the activity and the growth of the ammonium-oxidizing bacteria were repressed under drained and waterlogged conditions in soils with R. palustris , a condition that was attributed to a competitive ammonium uptake by its relatively fast growing roots. In the presence of R. acetosa , the activity and growth of the ammonium-oxidizing bacteria were inhibited under waterlogged, but not under drained, conditions. he growth and activity of nitrite-oxidizing bacteria in the absence of actively ammonium-oxidizing, nitrite-producing bacteria was likely due to organotrophic growth.     

1-Aminocyclopropane-1-Carboxylate Oxidase Activity Limits Ethylene Biosynthesis in Rumex palustris during Submergence

Submergence strongly stimulates petiole elongation in Rumex palustris, and ethylene accumulation initiates and maintains this response in submerged tissues. cDNAs from R. palustris corresponding to a 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene (RP-ACO1) were isolated from elongating petioles and used to study the expression of the corresponding gene. An increase in RP-ACO1 messenger was observed in the petioles and lamina of elongating leaves 2 h after the start of submergence. ACC oxidase enzyme activity was measured in homogenates of R. palustris shoots, and a relevant increase was observed within 12 h under water with a maximum after 24 h. We have shown previously that the ethylene production rate of submerged shoots does not increase significantly during the first 24 h of submergence (L.A.C.J. Voesenek, M. Banga, R. H. Thier, C.M. Mudde, F.M. Harren, G.W.M. Barendse, C.W.P.M. Blom [1993] Plant Physiol 103: 783-791), suggesting that under these conditions ACC oxidase activity is inhibited in vivo. We found evidence that this inhibition is caused by a reduction of oxygen levels. We hypothesize that an increased ACC oxidase enzyme concentration counterbalances the reduced enzyme activity caused by low oxygen concentration during submergence, thus sustaining ethylene production under these conditions. Therefore, ethylene biosynthesis seems to be limited at the level of ACC oxidase activity rather than by ACC synthase in R. palustris during submergence.     

New perspectives in flooding research: the use of shade avoidance and Arabidopsis thaliana

BACKGROUND: Complete submergence of Rumex palustris leads to hyponastic (upward) petiole growth followed by enhanced petiole elongation. Previous pharmacological experiments have provided insights into the signal transduction pathway leading to this combined 'escape' response. It will, however, be difficult to gain further knowledge using these methods. Consequently, new approaches are required. SCOPE: Here we propose that different environmental signals resulting in similar phenotypes can help to understand better the submergence response. In this review, we show that both ethylene and shade induce similar growth responses in R. palustris and Arabidopsis thaliana. We illustrate how this can be exploited to unravel novel signalling components in submergence-induced elongation growth. Furthermore, we illustrate the potential of arabidopsis as a useful model in submergence research based on similarities with submergence-tolerant species such as R. palustris and the molecular opportunities it presents. This is illustrated by examples of current work exploring this concept. CONCLUSIONS: Incorporating different model systems, such as arabidopsis and shade avoidance, into submergence research can be expected to create powerful tools to unravel signal transduction routes determining submergence tolerance.     

A kinetic analysis of hyponastic growth and petiole elongation upon ethylene exposure in Rumex palustris

Background and Aims

Complete submergence is an important stress factor for many terrestrial plants, and a limited number of species have evolved mechanisms to deal with these conditions. Rumex palustris is one such species and manages to outgrow the water, and thus restore contact with the atmosphere, through upward leaf growth (hyponasty) followed by strongly enhanced petiole elongation. These responses are initiated by the gaseous plant hormone ethylene, which accumulates inside plants due to physical entrapment. This study aimed to investigate the kinetics of ethylene-induced leaf hyponasty and petiole elongation.

Methods

Leaf hyponasty and petiole elongation was studied using a computerized digital camera set-up followed by image analyses. Linear variable displacement transducers were used for fine resolution monitoring and measurement of petiole growth rates.

Key Results

We show that submergence-induced hyponastic growth and petiole elongation in R. palustris can be mimicked by exposing plants to ethylene. The petiole elongation response to ethylene is shown to depend on the initial angle of the petiole. When petiole angles were artificially kept at 0°, rather than the natural angle of 35°, ethylene could not induce enhanced petiole elongation. This is very similar to submergence studies and confirms the idea that there are endogenous, angle-dependent signals that influence the petiole elongation response to ethylene.

Conclusions

Our data suggest that submergence and ethylene-induced hyponastic growth and enhanced petiole elongation responses in R. palustris are largely similar. However, there are some differences that may relate to the complexity of the submergence treatment as compared with an ethylene treatment.     

The role of oxygen in submergence-induced petiole elongation in Rumex palustris: in situ measurements of oxygen in petioles of intact plants using micro-electrodes

In a study on the mechanism of stimulated petiole elongation in submerged plants, oxygen concentrations in petioles of the flood-tolerant plant Rumex palustris were measured with micro-electrodes. Short-term submergence lowered petiole partial oxygen pressure to c . 19 kPa whereas prolonged submergence under continuous illumination depressed oxygen levels to c . 8–12 kPa after 24 h. Oxygen levels in petioles depended on the presence of the lamina, even in submerged conditions, and on available light. In darkness, petiole oxygen levels in submerged plants dropped quickly to values as low as 0.5–4 kPa. It is hypothesized that prolonged submergence in the light is accompanied by a decrease in carbon dioxide in the petiole. Submergence-enhanced petiolar elongation rate was compared with emergent plants. Peak daily elongation rates occurred at the end of the dark period in emergent plants, but in the middle of the light period in submerged plants. We suggest that this shift in daily elongation pattern is induced by dependence of growth on photosynthetically derived oxygen in submerged plants. Implications of reduced oxygen for ethylene production are raised. Levels of 1- aminocyclopropane-1-carboxylic acid synthase and 1-aminocyclopropane-1-carboxylic acid oxidase and ethylene sensitivity are cited as potential factors in hypoxia-induced ethylene release.