Petiole elongation inRumex species during submergence and ethylene exposure: The relative contributions of cell division and cell expansion

Submergence ofRumex crispus L. andR. palustris Sm. stimulates elongation of the youngest petiole. InR. palustris this response can be mimicked partially by exposure to exogenous ethylene. In both species, petiole elongation induced by ethylene and/or submergence is distributed nearly equally over the whole petiole length and is almost completely attributable to increased cell expansion. InR. acetosa L., extension of the youngest petiole is inhibited by submergence of the whole plant. The strongest growth inhibition within the youngest petiole was observed in the most distal parts and is probably the result of reduced cell expansion.     

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.     

Ethylene enhances gibberellin levels and petiole sensitivity in flooding-tolerant Rumex palustris but not in flooding-intolerant R. acetosa

The role of gibberellin (GA) and ethylene in submergence-induced petiole elongation was studied in two species of the genus Rumex. Analysis of endogenous GAs in the flooding-tolerant Rumex palustris Sm. and the intolerant Rumex acetosa L. by gas chromatography-mass spectrometry showed for both species the presence of GA₁, GA₄, GA₉, GA₁₉, GA₂₀ and GA₅₃. Gas chromatography-mass spectrometry analysis of R. palustris petiole tissue of submerged plants showed an increase in levels of 13-OH GAs, especially GA₁, compared with drained plants. This effect could be mimicked by application of 5 μL L⁻¹ ethylene. In R. acetosa, no differences between levels of GAs in drained or submerged plants were found. In R. palustris, both submergence and ethylene treatment sensitized petioles to exogenous gibberellic acid (GA₃). In R. acetosa the effect was opposite, i.e. submergence and ethylene de-sensitized petioles to GA₃. Our results demonstrate the dual effect of ethylene in the submergence response related to flooding tolerance, i.e. in the flooding-tolerant R. palustris ethylene causes an increased concentration of and sensitivity to GA with respect to petiole elongation while in the intolerant R. acetosa ethylene reduces growth independent of GAs. Received: 5 November 1996 / Accepted: 8 February 1997     

Is elongation-induced leaf emergence beneficial for submerged Rumex species?

Background and Aims

Plant species from various taxa ‘escape’ from low oxygen conditions associated with submergence by a suite of traits collectively called the low oxygen escape syndrome (LOES). The expression of these traits is associated with costs and benefits. Thus far, remarkably few studies have dealt with the expected benefits of the LOES.

Methods

Young plants were fully submerged at initial depths of 450 mm (deep) or 150–240 mm (shallow). Rumex palustris leaf tips emerged from the shallow flooding within a few days, whereas a slight lowering of shallow flooding was required to expose R. acetosa leaf tips to the atmosphere. Shoot biomass and petiole porosity were measured for all species, and treatments and data from the deep and shallow submergence treatments were compared with non-flooded controls.

Key Results

R. palustris is characterized by submergence-induced enhanced petiole elongation. R. acetosa lacked this growth response. Upon leaf tip emergence, R. palustris increased its biomass, whereas R. acetosa did not. Furthermore, petiole porosity in R. palustris was twice as high as in R. acetosa.

Conclusions

Leaf emergence restores gas exchange between roots and the atmosphere in R. palustris. This occurs to a much lesser extent in R. acetosa and is attributable to its lower petiole porosity and therefore limited internal gas transport. Leaf emergence resulting from fast petiole elongation appears to benefit biomass accumulation if these plants contain sufficient aerenchyma in petioles and roots to facilitate internal gas exchange.Key words: Submergence, emergence, enhanced shoot elongation, porosity, aerenchyma, Rumex, cost–benefit analysis, phenotypic plasticity     

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.     

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.     

Sensitivity to ethylene: the key factor in submergence-induced shoot elongation of Rumex

Rosettes of flooding-resistant Rumex palustris plants show a submergence-induced stimulation of elongation, which is confined to the petioles of young leaves. This response increases the probability of survival. It is induced by ethylene that accumulates in submerged tissues. Flooding-intolerant Rumex acetosella plants do not show this response. We investigated whether differences in shoot elongation between the species, between old and young leaves and between the petiole and leaf blade of a R. palustris plant result from differences in internal ethylene concentration or in sensitivity to the gas. Concentrations of free and conjugated ACC in petioles and leaf blades of R. palustris indicated that ethylene is synthesized throughout the submerged shoot, although production rates varied locally. Nevertheless, no differences in ethylene concentration were found between submerged leaves of various ages. In contrast, dose-response curves showed that only elongation of young petioles of R. palustris was sensitive to ethylene. In R. acetosella, elongation of all leaves was insensitive to ethylene. We conclude that variation in ethylene sensitivity rather than content explains the differences in submergence-induced shoot elongation between the two Rumex species and between leaves of R. palustris.     

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.     

Enhancement of auxin sensitivity in Ranunculus sceleratus by ethylene: A mechanism to escape from hypoxia under temporary submergence

We analyzed auxin-induced and ethylene-enhanced elongation of petiole segments in Ranunculus sceleratus L. The early time course of elongation in petiolar segments was monitored with a computer-based video digitizer system. The application of ethylene-releasing ethrel slightly increased the elongation rate in the absence of IAA. When IAA alone was applied, elongation increased after a latent period of approximately 30 min. Maximal elongation rate was attained immediately after the latent period, and then the stabilized steady rate was recorded. During this phase, addition of ethrel strongly increased the elongation rate after a period of approximately 18 min. Although ethrel could acidify the growth medium, only a small part of the enhanced elongation was due to an acid-growth effect. Most of the growth stimulation was auxin-dependent and must be ascribed to the presence of ethylene. In the presence of ethrel, the log-concentration-response curve of IAA appeared to be shifted to the left. This kinetic analysis indicates an increase, due to ethylene, in the sensitivity of the R. sceleratus petiole to auxin, which results in inducing rapid growth to escape from hypoxia under temporary submergence.     

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.     

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.     

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.     

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.     

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 Promotes Elongation Growth and Auxin Promotes Radial Growth in Ranunculus sceleratus Petioles

Submergence induces elongation in the petioles of Ranunculus sceleratus L., after a rise in endogenous ethylene levels in the tissue. Petioles of isolated leaves also elongate 100% in 24 hours when treated with ethylene gas, without a change in the radius. Application of silver thiosulfate, aminoethoxyvinylglycine (AVG), abscisic acid (ABA), or methyl jasmonate inhibits this elongation response. Gibberellic acid treatment promotes ethylene-induced elongation, without an effect on the radius. Indoelastic acid (IAA) induces radial growth in the petioles, irrespective of the presence or absence of added ethylene. High concentrations of IAA will also induce elongation growth, but this is largely due to auxin-induced ethylene synthesis; treatment with silver thiosulfate, AVG, ABA, or methyl jasmonate inhibit this auxin-promoted elongation growth. However, the radial growth induced by IAA is not affected by gibberellic acid, and not specifically inhibited by ABA, methyl jasmonate, silver thiosulfate, or AVG. These results support the idea that petiole cell elongation during “accommodation growth” can be separated from radial expansion. The radial expansion may well be regulated by IAA. However, effects of high levels of IAA are probably anomalous, since they do not mimic normal developmental patterns.     

Plant movement. Submergence-induced petiole elongation in Rumex palustris depends on hyponastic growth

The submergence-tolerant species Rumex palustris (Sm.) responds to complete submergence by an increase in petiole angle with the horizontal. This hyponastic growth, in combination with stimulated elongation of the petiole, can bring the leaf tips above the water surface, thus restoring gas exchange and enabling survival. Using a computerized digital camera set-up the kinetics of this hyponastic petiole movement and stimulated petiole elongation were studied. The hyponastic growth is a relatively rapid process that starts after a lag phase of 1.5 to 3 h and is completed after 6 to 7 h. The kinetics of hyponastic growth depend on the initial angle of the petiole at the time of submergence, a factor showing considerable seasonal variation. For example, lower petiole angles at the time of submergence result in a shorter lag phase for hyponastic growth. This dependency of the hyponastic growth kinetics can be mimicked by experimentally manipulating the petiole angle at the time of submergence. Stimulated petiole elongation in response to complete submergence also shows kinetics that are dependent on the petiole angle at the time of submergence, with lower initial petiole angles resulting in a longer lag phase for petiole elongation. Angle manipulation experiments show that stimulated petiole elongation can only start when the petiole has reached an angle of 40 degrees to 50 degrees. The petiole can reach this "critical angle" for stimulated petiole elongation by the process of hyponastic growth. This research shows a functional dependency of one response to submergence in R. palustris (stimulated petiole elongation) on another response (hyponastic petiole growth), because petiole elongation can only contribute to the leaf reaching the water surface when the petiole has a more or less upright position.     

Petiole Growth in Ranunculus sceleratus L.: Ethylene Synthesis and Submergence

Petioles of the celery-leaved buttercup (Ranunculus sceleratusL.) elongate in response to treatment with ethylene in air whenthe leaf blades are attached. An enhanced rate of elongationgrowth also occurs when the leaves are submerged. Submergencecauses an increase in extractable ethylene gas within the tissues,and these levels appear to approach those required to saturatethe ethylene-promoted elongation growth response. Coincidentwith a rise in ethylene in the tissues is a dramatic increasein the level of I-aminocyclopropane-1-carboxylic acid (ACC),the precursor of ethylene. Both the petiole and leaf blade tissueshave a similar capacity to evolve ethylene in the presence ofadded ACC. However, in air the leaf blade evolves more ethylenefrom endogenous resources than the petiole. The simultaneousincreases in ethylene and ACC levels in submerged tissues areconsidered in terms of the low diffusivity of ethylene in water,the ‘autocatalytic’ effect of ethylene on ethylenebiosynthesis and the rôle of both carbon dioxide and oxygenfluxes in ethylene metabolism of submerged tissues. Ranunculus sceleratus, celery-leaved buttercup, petiole growth, submergence, ethylene metabolism