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.     

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.     

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.     

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.     

Ethylene-induced hyponastic growth in Arabidopsis thaliana is controlled by ERECTA

Plants can respond quickly and profoundly to detrimental changes in their environment. For example, Arabidopsis thaliana can induce an upward leaf movement response through differential petiole growth (hyponastic growth) to outgrow complete submergence. This response is induced by accumulation of the phytohormone ethylene in the plant. Currently, only limited information is available on how this response is molecularly controlled. In this study, we utilized quantitative trait loci (QTL) analysis of natural genetic variation among Arabidopsis accessions to isolate novel factors controlling constitutive petiole angles and ethylene-induced hyponastic growth. Analysis of mutants in various backgrounds and complementation analysis of naturally occurring mutant accessions provided evidence that the leucin-rich repeat receptor-like Ser/Thr kinase gene, ERECTA , controls ethylene-induced hyponastic growth. Moreover, ERECTA controls leaf positioning in the absence of ethylene treatment. Our data demonstrate that this is not due to altered ethylene production or sensitivity.     

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.     

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.     

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.     

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.     

On the Relevance and Control of Leaf Angle

Plants can have constitutive leaf angles that are fixed and do not vary much among different growth environments. Several species, however, have the ability to actively adjust their leaf angles. Active leaf repositioning can be functional in avoiding detrimental environmental conditions, such as avoidance of heat stress and complete submergence, or can be, for example, utilized to maximize carbon gain by positioning the leaves relative to the incoming radiation. In recent years, major advances have been made in the understanding of the molecular mechanisms, and the underlying hormonal regulation of a particular type of leaf movement: hyponastic growth. This differential petiole growth-driven upward leaf movement is now relatively well understood in model systems such as Rumex palustris and Arabidopsis thaliana. In the first part of this review we will discuss the functional consequences of leaf orientation for plant performance. Next, we will consider hyponastic growth and describe how exploitation of natural (genetic) variation can be instrumental in studying the relevance and control of leaf positioning.     

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 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.     

Abscisic acid antagonizes ethylene-induced hyponastic growth in Arabidopsis

Ethylene induces enhanced differential growth in petioles of Arabidopsis (Arabidopsis thaliana), resulting in an upward movement of the leaf blades (hyponastic growth). The amplitude of this effect differs between accessions, with Columbia-0 (Col-0) showing a large response, while in Landsberg erecta (Ler), hyponastic growth is minimal. Abscisic acid (ABA) was found to act as an inhibitory factor of this response in both accessions, but the relationship between ethylene and ABA differed between the two; the ability of ABA to inhibit ethylene-induced hyponasty was significantly more pronounced in Col-0. Mutations in ABI1 or ABI3 induced a strong ethylene-regulated hyponastic growth in the less responsive accession Ler, while the response was abolished in the ABA-hypersensitive era1 in Col-0. Modifications in ABA levels altered petiole angles in the absence of applied ethylene, indicating that ABA influences petiole angles also independently from ethylene. A model is proposed whereby the negative effect of ABA on hyponastic growth is overcome by ethylene in Col-0 but not in Ler. However, when ABA signaling is artificially released in Ler, this regulatory mechanism is bypassed, resulting in a strong hyponastic response in this accession.     

Genomic fingerprinting of Frankia strains by PCR-based techniques. Assessment of a primer based on the sequence of 16S rRNA gene of Escherichia coli

Submergence stimulates elongation of the leaves of Rumex palustris and under laboratory conditions the maximum final leaf length (of plants up to 7 weeks old) was obtained within a 9 day period. This elongation response, mainly determined by petiole elongation, depends on the availability of storage compounds and developmental stage of a leaf. A starch accumulating tap root and mature leaves and petioles were found to supply elongating leaves with substrates for polysaccharide synthesis in expanding cell walls. Changes in the composition of cell wall polysaccharides of elongated petioles suggest a substantial cell wall metabolism during cell extension. Reduced starch levels or removal of mature leaves caused a substantial limitation of submerged leaf growth. From the 5th leaf onward enough reserves were available to perform submerged leaf growth from early developmental stages. Very young petioles had a limited capacity to elongate. In slightly older petioles submergence resulted in the longest final leaf lengths and these values gradually decreased when submergence was started at more mature developmental stages. Submerged leaf growth is mainly a matter of petiole elongation in which cell elongation has a concurrent synthesis of xylem elements in the vascular tissue. Mature petioles still elongated (when submerged) by cell and tissue elongation only: the annular tracheary elements stretched enabling up to 70% petiole elongation.     

Ethylene-induced differential growth of petioles in Arabidopsis. Analyzing natural variation, response kinetics, and regulation

Plants can reorient their organs in response to changes in environmental conditions. In some species, ethylene can induce resource-directed growth by stimulating a more vertical orientation of the petioles (hyponasty) and enhanced elongation. In this study on Arabidopsis (Arabidopsis thaliana), we show significant natural variation in ethylene-induced petiole elongation and hyponastic growth. This hyponastic growth was rapidly induced and also reversible because the petioles returned to normal after ethylene withdrawal. To unravel the mechanisms behind the natural variation, two contrasting accessions in ethylene-induced hyponasty were studied in detail. Columbia-0 showed a strong hyponastic response to ethylene, whereas this response was almost absent in Landsberg erecta (Ler). To test whether Ler is capable of showing hyponastic growth at all, several signals were applied. From all the signals applied, only spectrally neutral shade (20 micromol m(-2) s(-1)) could induce a strong hyponastic response in Ler. Therefore, Ler has the capacity for hyponastic growth. Furthermore, the lack of ethylene-induced hyponastic growth in Ler is not the result of already-saturating ethylene production rates or insensitivity to ethylene, as an ethylene-responsive gene was up-regulated upon ethylene treatment in the petioles. Therefore, we conclude that Ler is missing an essential component between the primary ethylene signal transduction chain and a downstream part of the hyponastic growth signal transduction pathway.     

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.