• Earlier work on the submergence-tolerant species Rumex palustris revealed that leaf anatomical and morphological changes induced by submergence enhance underwater gas exchange considerably. Here, the hypothesis is tested that these plastic responses are typical properties of submergence-tolerant species.
• Submergence-induced plasticity in leaf mass area (LMA) and leaf, cell wall and cuticle thickness was investigated in nine plant species differing considerably in tolerance to complete submergence. The functionality of the responses for underwater gas exchange was evaluated by recording oxygen partial pressures inside the petioles when plants were submerged.
• Acclimation to submergence resulted in a decrease in all leaf parameters, including cuticle thickness, in all species irrespective of flooding tolerance. Consequently, internal oxygen partial pressures (pO2) increased significantly in all species until values were close to air saturation. Only in nonacclimated leaves in darkness did intolerant species have a significantly lower pO2 than tolerant species.
• These results suggest that submergence-induced leaf plasticity, albeit a prerequisite for underwater survival, does not discriminate tolerant from intolerant species. It is hypothesized that these plastic leaf responses may be induced in all species by several signals present during submergence; for example, low LMA may be a response to low photosynthate concentrations and a thin cuticle may be a response to high relative humidity.
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Recent investigations have identified a novel aspect of submergence-induced shoot plasticity. A case study with a model plant for submergence studies, Rumex palustris, showed a suite of changes at the leaf level upon complete submergence. The thickness of the lamina, cuticle and epidermal cell walls was lower in acclimated leaves that had developed under water compared with nonacclimated leaves, as was the ratio between leaf mass and leaf area (Mommer et al., 2005b). This plasticity resulted in increased gas exchange between leaves and water column by more than tenfold after 10 d of submergence (Mommer et al., 2006b), thereby increasing the oxygen status of the shoot up to near air-saturation (Mommer et al., 2004). Our current study investigates whether this leaf plasticity of R. palustris is species specific or is more generally observed in submergence-tolerant plant species.
Comparable, yet more extreme examples of such leaf plasticity are known from several truly aquatic plant species, in which this plasticity is aptly termed ‘heterophylly’ (Sculthorpe, 1967). Underwater, a different leaf morphology develops compared with that of aerial leaves, again resulting in superior underwater gas exchange capacities (Nielsen, 1993; Frost-Christensen & Sand-Jensen, 1995; Bruni et al., 1996; Sand-Jensen & Frost-Christensen, 1998; Robe & Griffiths, 2000; Wells & Pigliucci, 2000; Frost-Christensen et al., 2003). The similar responses of aquatic and wetland species suggest that this leaf acclimation may be a typical property of flooding-tolerant plant species, and one not likely to be expressed in intolerant species. Alternatively, the submergence-induced plasticity of the leaf may not necessarily be an adaptation to submergence specifically, but might be induced by different signals present during submergence such as low light, low photosynthate concentrations and high relative humidity. In such a case, we would expect submergence-induced leaf plasticity also to occur in intolerant plant species.
Here, the hypothesis was tested that leaf plasticity in response to complete submergence is related to the flooding tolerance of a species, as confirmed for other submergence-induced traits such as increased plant height, increased aerenchyma content and reduced leaf mass area (LMA; Mommer et al., 2006a). To this aim, an oxygen microelectrode study was performed, to evaluate underwater gas exchange in a set of nine species differing in flooding tolerance. Internal oxygen partial pressure (pO2) was measured in the petioles as affected by oxygen influx from the surrounding water and from underwater photosynthesis. These measurements were combined with in-depth investigations into the morphological and anatomical acclimations that have been recently identified to regulate underwater gas exchange (Mommer et al., 2005b).
Materials and Methods
Species selection and plant material
The relationship was investigated between leaf anatomy and underwater gas exchange in nine species that commonly occur in the river floodplain grasslands of the river Rhine in the Netherlands (Table 1) (Sýkora et al., 1988; van Eck et al., 2005). The species selection comprises five different dicotyledonous families, within which a tolerant and an intolerant species were chosen in order to avoid phylogenetic effects (cf. Jansen et al., 2005; Mommer et al., 2006a).
Table 1. List of species used in the experiments, including degree of submergence tolerance and hydrological characteristics of their habitats
The degree of tolerance of the selected species had experimentally been quantified in earlier work by determining the median survival time (LT50) during complete submergence in low light conditions (30 µmol m−2 s−1; Mommer et al., 2006a). Although this survival assessment under controlled experimental conditions may in principle overestimate survival under true flooding in the field, van Eck et al. (2004) and Voesenek et al. (2004) convincingly showed that these experimental survival times correlate well with the species distribution in the field. The so-called tolerant species (with a group average LT50 of 150 d) inhabit the wet grasslands and mud flats near the river Rhine, where the average flooding duration is often > 100 d yr−1 (classes C4, C7 and C8 in Voesenek et al., 2004). The so-called intolerant species (with a group average LT50 of 28 d) originate from the dry grassland communities on the high elevated dikes and river dunes, where flooding is of very short duration (on average < 2 d yr−1) or absent (classes C1 and C2 in Voesenek et al., 2004). Within the two tolerance classes, there was variation in survival time among species (Mommer et al., 2006a). However, within a species pair from a family, both a tolerant and an intolerant species were selected, with a very pronounced survival time difference (cf. Mommer et al., 2006a). Degrees of tolerance were thus not used as absolute values, but should be interpreted as relative between the two classes. In conclusion, species classification is based on species distribution in the field and corresponding experimental survival rates.
All plant species, except Potentilla reptans, were grown from seeds. Seeds were collected from populations in the floodplains near Nijmegen, the Netherlands, except, because of insufficient seed set, Potentilla verna, which originated from a population in the botanical garden of Baldwinstein, Germany. The seeds were germinated on moistened filter paper in Petri dishes (photosynthetic photon flux density (PPFD) was 20 µmol m−2 s−1, with a day:night cycle of 16 : 8 h and temperature 20 : 10°C) for 14 d. Then, the seedlings were transplanted into pots of size 6 × 6 × 8 cm containing a sieved sand:potting soil mixture (1 : 1 volume/volume (v/v)), and grown for another 32 d in a growth chamber (PPFD at leaf level 250 µmol m−2 s−1 (SON-T plus 600 W and TLD Reflex 36W/840R; Philips, Eindhoven, the Netherlands); day:night cycle 16 : 8 h; temperature 20°C). Plants of P. reptans originated from cuttings of populations grown in the common garden of the Radboud University Nijmegen, but were originally collected in the floodplains near Utrecht (the Netherlands). The cuttings were grown for 21 d under the same conditions in a climate room as described above. All plants were watered with half-strength Hoagland's nutrient solution three times a week. The position of the pots was changed regularly to homogenize growth conditions among replicate plants.
To investigate the effect of submergence acclimation on underwater gas exchange and leaf anatomy, plants were subjected to either a flooding or drained (control) treatment for 10–14 d, until the plants had developed at least one mature leaf during the treatment. Plants in the flooded treatment were completely submerged in polyethylene basins (volume 340 l, depth of water column 55 cm). The basins were filled with tap water, which was circulated through a filter system with a flow rate of 1.5 l min−1. The CO2 and O2 pressures of the water were air-saturated (at pH 8.3), i.e. 15 µm (equivalent to ambient air conditions: 350 Pa) and 21 kPa, respectively. Plants in the drained treatment were placed in similar basins that were not filled with water. All basins were placed in a growth chamber with similar conditions as described in the previous section. Light conditions in the basins at leaf level were 110 µmol m−2 s−1 PPFD (SON-T plus 600 W and TLD Reflex 36 W/840R; Philips).
The leaves that developed in the submergence treatment will be referred to as ‘aquatic’ leaves, and the leaves that developed in the drained treatment will be referred to as ‘terrestrial’ leaves (cf. Mommer et al., 2005b). All measurements were performed on the youngest full-grown leaf.
Oxygen microelectrode measurements
Oxygen microelectrode measurements were performed on the petioles of plants that were placed in an aquarium (volume 9 l) filled with tap water, within half an hour of their removal from their respective treatments. Submerged plants were kept submerged during transport. The plants, including intact root systems with soil in a plastic bag, were mounted horizontally in the aquarium on a plateau. The petiole of the youngest full-grown leaf was kept in place on the plateau with thin rubber bands, in such a way that the petiole at 3 mm below the lamina was fixed. At this position, microelectrodes (tip diameter 25 µm) were inserted to a depth of 25% of the diameter of the petiole. The microelectrode was inserted at the upper end of the petiole, because this point is close to the major source of oxygen (i.e. the lamina) and allows comparisons among leaf types and species independently from putative differences in petiole porosity. Insertion of the microelectrode into the lamina itself was not feasible, because several species developed leaves that were too thin or fragile for such a measurement. Care was taken that water circulation was maintained around all leaves, including the target leaf, by bubbling the water in the aquaria with pressurized air containing slightly higher CO2 concentrations than ambient (470 Pa) and 21 kPa oxygen, respectively. This resulted in a flow velocity of the water of 2–5 cm s−1, which minimized the formation of boundary layers around the leaves.
Oxygen microelectrodes (OX25; Unisense, Aarhus, Denmark; Revsbech, 1989) were used to monitor the oxygen partial pressure (pO2) within the petioles of the plant (Mommer et al., 2004). Data were logged from a picoamp meter (PA8000; Unisense) every 15 s with an a/d converter (AD16; PicoTech, St Neots, UK). Electrode signals were converted to oxygen partial pressures using individual calibrations between 0 and 100% of air saturation before and after each series of measurements. The measurements were performed in both complete darkness, when only passive diffusion of oxygen from the water column into the lamina occurs, and in light (PPFD 180–200 µmol m−2 s−1; SON-T plus 600 W and TLD Reflex 36 W/840R; Philips), when photosynthesis may generate additional oxygen.
The microelectrode measurements were performed in a growth chamber at 20°C. To keep the temperature constant, the aquarium in which the measurements were performed was placed in a larger aquarium (volume 18 l) that contained thermostat-controlled circulating water at 20°C. All measurements were replicated three to five times.
Leaf anatomical parameters
Light microscopy and transmission electron microscopy were used to investigate leaf anatomy. Samples of the lamina were taken from the upper one-third of the leaf within 5 s of removal of the plants from their respective treatment. Fixation of the samples occurred in 2% glutaraldehyde in 0.1 m phosphate buffer at pH 7.2 for 2 h at 20°C, and postfixation occurred in 1% (weight/volume (w/v)) osmium tetroxide in the same buffer for 1 h. The leaf samples were dehydrated through an ethanol series with steps of 10% and subsequently, via a propylene oxide step, embedded in Spurr's resin (Spurr, 1969). Sections of 1 µm were stained with toludine blue (0.1% in 1% borax) and viewed with a Leitz Ortoplan light microscope (Leitz, Wetzlar, Germany). Thinner sections (20 nm) were poststained with uranyl acetate and lead citrate according to standard procedures and viewed with a transmission electron microscope (JEOL JEM 100CX II; Jeol, Tokyo, Japan). The thickness of the leaves (9 counts within each leaf sample; n = 2) and of the outer cell walls of epidermal cells and cuticles (15 counts within each leaf sample; n = 1) of the adaxial side of the lamina were measured with digital software (ImageJ; RSB, Bethesda, MD, USA). Regions of the leaf that included mid-veins were excluded from analyses.
LMA, a parameter that expresses the biomass investment per leaf area, was recorded from the same leaf as used for the microelectrode measurements. LMA was calculated from the lamina area, measured with a leaf area meter (LI 3000; Li-Cor, Lincoln, NE, USA), and the leaf dry weight, determined after drying for at least 48 h at 80°C.
The tissue porosity (measured as the relative volume of air spaces) within both the lamina and the petiole of the leaf is positively correlated with the capacity of the plant for within-plant gas diffusion and was recorded using the microbalance method as described in Visser & Bögemann (2003) after a light period of c. 3 h. There were no apparent gas films present around the leaves of these species.
The effects of submergence acclimation on internal oxygen partial pressure and plant parameter expression within species were assessed with a repeated measures analysis of variance (ANOVA) (sas glm procedure; SAS Institute, Cary, NC, USA), considering each species as an independent subject. The degree of acclimation (leaf type, terrestrial or aquatic) was considered as a within-subject effect and flooding tolerance (tolerant (T) or intolerant (I)) as a between-subject effect. Transformations were performed on some parameters so that all data sets met the assumptions of repeated measures ANOVA. The species Salvia pratensis was not included in these analyses, because it did not develop aquatic leaves in the submergence treatment.
Correlations among leaf anatomical parameters were assessed with Pearson's correlation analysis (sas corr procedure). To relate leaf anatomical changes to underwater gas exchange, correlations of the leaf anatomical parameters with pO2 in darkness were tested. Analyses were performed with species means. All statistical analyses were conducted with sas 9.1 (SAS Institute).
pO2 is largely similar among species with different degrees of submergence tolerance
We observed a beneficial effect of acclimation to submergence on internal oxygen status of the shoot in all species tested, as indicated by a significant increase of internal oxygen partial pressures (pO2) in aquatic compared with terrestrial upper petioles in both dark and light conditions (Fig. 1a,b; significant leaf type effect).
Terrestrial leaves of intolerant species had significantly lower pO2 in their upper petiole underwater in darkness than tolerant species (Fig. 1a; significant tolerance effect). This disadvantage in terms of pO2 in the intolerant species, however, disappeared after acclimation. After acclimation, the two groups of species had similar oxygen status, as indicated by the significant interaction between leaf type and degree of tolerance (Fig. 1; resulting in a significant interaction term). In light conditions, when photosynthesis occurred, pO2 was higher than in darkness and close to the ambient oxygen partial pressures in the water layer. In the light, we did not observe significant differences between species with different degrees of tolerance, either before or after acclimation. Thus, intolerant species have surprisingly similar pO2 in the submerged upper petiole compared with tolerant species, except for their nonacclimated terrestrial leaves in darkness.
Leaf anatomical responses to submergence are general across species
All investigated leaf anatomical traits – LMA, leaf thickness, and cell wall and cuticle thickness of the adaxial side of the leaf – showed significant reductions in the aquatic compared with the terrestrial leaves (Figs 2, 3; significant leaf type effect). The aquatic leaves were thinner, had thinner outer cell walls and cuticles and had a larger surface area per biomass ratio than their aerial counterparts.
The magnitude of the leaf trait responses was not lower in intolerant than in tolerant species. Intolerant species even showed more pronounced responses in epidermal cell wall thickness (Fig. 3c; significant interaction term), which follows from the fact that tolerant species already had constitutively thin cell walls.
One of the most tolerant species in our experiment, Oenanthe aquatica, did not follow the general pattern as it did not express any plasticity in leaf thickness or adaxial cell wall or cuticle thickness. This is surprising at first sight, but this species may already be inherently suited to an aquatic life (Figs 1, 2, 3).
We observed overall negative significant correlations between pO2 and all four leaf anatomical characteristics, indicating that thinner leaves, thinner cell walls and cuticles and less biomass per leaf area enhance the internal pO2 (Table 2a). Correlation analyses among leaf anatomical parameters of both terrestrial and aquatic leaves separately revealed that the thickness of the cuticle was positively correlated with LMA (Table 2b).
Table 2. Significance of Pearson's correlations (a) between leaf traits and pO2 in darkness (n = 17) and (b) among leaf anatomical parameters of different leaf types
Although the less tolerant species showed a greatly reduced porosity of the lamina when submerged, this difference was not statistically significant in comparison with the tolerant species (no significant leaf type × flooding tolerance effect). This is probably attributable to the apparent decrease of porosity in the tolerant O. aquatica (Fig. 2, Table 3). However, we observed that the leaves formed by this species underwater were extremely sensitive to desiccation during the porosity assay, which may have led to erratic values. Gas space content in the petiole showed a marginally significant increase upon submergence, with the flooding-tolerant species tending to have higher porosity contents (marginal effect of tolerance; Table 3).
Table 3. Internal gas spaces in the lamina and petiole of submergence-acclimated (Aqu.) and nonacclimated (Ter.) leaves of different species and the outcome of the statistical analyses (repeated measures analysis of variance)
Gas volume in lamina (%)
Gas volume in petiole (%)
The negative value of gas volume in O. aquatica indicates gas volumes close to zero (see Fig. 2); however, for this species we cannot rule out the possibility that the fast desiccation of the aquatic leaves once they had been taken out of the water affected the accuracy of measurements.
Data are mean ± standard error; n = 6–10. $, P < 0.1; *, P < 0.05; ns, not significant. T and I (‘tolerant’ and ‘intolerant’) indicate the degree of tolerance of a species (see Table 1). Salvia pratensis was not included in the analysis as it did not form aquatic leaves.
This experiment shows that submergence-induced leaf acclimation may be a more general phenomenon than hitherto believed, as we found similar reductions of LMA, leaf thickness, cell wall and cuticle thickness and outer cell wall thickness in nine terrestrial species that differed substantially in flooding tolerance. This submergence-induced leaf plasticity resulted in increased oxygen partial pressures (pO2) in the upper petioles, and thus probably in most of the shoot in all species. Therefore, the ability to induce plastic leaf responses to submergence appears not to be related to flooding tolerance. Only in darkness immediately after submergence did flooding-intolerant plant species have significantly lower pO2 in their petioles than tolerant species, suggesting inherent superior leaf characteristics to cope with submerged conditions in the tolerant species.
Leaf plasticity does not determine flooding tolerance
The nine species tested here showed leaf acclimation in response to submergence and, consequently, increased pO2 of the submerged petiole after leaf acclimation. In light conditions, pO2 was close to air saturation or even slightly supersaturated, irrespective of the degree of acclimation of the shoot, which indicates that leaf plasticity in response to submergence is not a key feature determining flooding tolerance. This was also true when the light intensity during the microelectrode measurement was very low (20 µmol m−2 s−1; data not shown). The finding that pO2 in the upper petioles of acclimated intolerant species was almost as high as in the acclimated tolerant species could in principle also (partly) result from reduced respiratory demands in dying tissues of intolerant species, rather than from increased influx of CO2 (in the light) or O2 (in dark) as in tolerant species. However, this would be inconsistent with the substantial changes in leaf anatomy in the intolerant species, which were very similar to those in the tolerant species. It is highly unlikely that these changes would not affect gas fluxes in intolerant species but would do so in tolerant species.
Because our conclusions have been based on measurements of pO2 in the upper petiole, and leaf types and species may differ in both petiole length and aerenchyma content in the petiole, it must be considered that different gradients of pO2 may develop between shoot and roots. It will therefore be an interesting exercise in future experiments to obtain more information on these gradients and their importance in explaining potential differences in root survival. This will help to further elucidate the role of shoot acclimation in terrestrial plant survival underwater.
In contrast to light conditions, flooding-tolerant species in darkness had significantly higher pO2 than intolerant species, particularly in nonacclimated leaves (Fig. 1), suggesting an inherent advantage of the leaf characteristics of flooding-tolerant species. This initial advantage may be crucial for survival underwater as the development of acclimated leaves underwater takes several days, and flooding conditions can be very turbid, particularly during the first few days (Vervuren et al., 2003). Leaf longevity of terrestrial leaves is shorter in intolerant than in tolerant species (Mommer et al., 2006a), and, moreover, the production of aquatic leaves is considerably lower in intolerant species than in tolerant species (Mommer & Visser, 2005). For example, the most intolerant species tested in this study, S. pratensis (Mommer et al., 2006a), did not even form aquatic leaves. However, it is unclear whether this is the cause or the effect of intolerance.
The aim of this study was not to explain the differences in tolerance to complete submergence among species. However, our data seem to rule out leaf anatomy as a causal factor to discriminate tolerant from intolerant species. Rather, submergence-induced leaf plasticity should be considered a prerequisite for underwater survival. Alternatively, an important factor determining submergence tolerance could be differences in internal aeration that exist between flooding-tolerant and -intolerant species, as this determines the diffusion of oxygen from the leaves via the petioles/stems into the roots (Voesenek et al., 2006). Consistently, we showed in a previous study that the porosity of the petiole is positively correlated with plant survival during complete submergence (Mommer et al., 2006a), and this finding is confirmed in the current study, where tolerant species tended to have a higher porosity content in the petioles (Table 3). Of course, internal aeration will only be functional if sufficient oxygen can enter the lamina (directly or indirectly as a photosynthetic consequence of CO2 diffusion in the light) to diffuse through the rest of the plant. We therefore suggest enhanced gas exchange underwater to be a prerequisite for survival, whereas between-species variation in internal aeration may explain the variation in submergence tolerance between species.
Different traits, different mechanistic backgrounds?
The generality of the leaf responses to submergence in all nine species tested may suggest that these responses are not necessarily an adaptation to submergence specifically, as the intolerant species have not been subjected to recent selection for submergence responses. The general leaf plasticity is probably better understood when evaluated as a suite of various traits that have different mechanistic origins, as they might be components of responses to environmental factors other than submergence, such as shade or high relative humidity.
The similar decreases of LMA in very different environments – underwater and in shade – are, however, intriguing and prompted us to speculate about the regulation of this common plant trait. Light intensity itself cannot be the trigger of submergence-induced LMA reduction as light intensity was similar between the submerged and drained treatments. However, both in shade and underwater, rates of photosynthesis are much reduced because of low light and low CO2 availability, respectively. Possibly, the reduced rates of photosynthesis directly or indirectly induce reduced LMA. This hypothesis is strengthened by work on transgenic tobacco (Nicotiana tabacum) plants with reduced Rubisco concentrations, leading to a 50% reduction in photosynthesis compared with wild-type controls (Evans et al., 1994), and an 18% reduction in LMA.
Although LMA based on leaf samples did not correlate with underwater gas exchange, whole-plant LMA was shown to be positively correlated with flooding tolerance (Mommer et al., 2006a). This observation might be related to the fact that whole-plant LMA also comprises leaf veins and petioles, and lower investments in these leaf components result in lower construction and maintenance requirements, ultimately leading to benefits underwater, where the energy status is severely reduced.
An important difference in the acclimation responses to submergence and low light is the plasticity of the cuticle layer. Aquatic leaves of all species tested had a large reduction in cuticle thickness of at least 40%, and even up to 90% (similar to the data obtained by Frost-Christensen et al., 2003 for aquatic species). These are values that are unprecedented for shade treatments, where no reduction (Hauke & Schreiber, 1998) or at best a mild tendency for a reduction (Baltzer & Thomas, 2005) in cuticle thickness has been observed. We therefore suggest that the decrease in cuticle thickness is an important indicator for increased gas exchange properties underwater. Cuticle formation may be regulated by relative humidity, amongst other factors, as was recently shown for Brassica oleracea, where the total amount of cuticle wax decreased by 50% after transition from control (40–75%) to high (98%) relative humidity (Koch et al., 2006). Submerged conditions are, of course, at the extreme high end of the relative humidity scale, potentially leading to the large decreases in cuticle thickness that we observed underwater.
We found that leaf acclimation responses to submergence have a beneficial impact on the pO2 of the shoot. However, because leaf acclimation seems similar in species that are tolerant and intolerant to submergence, it does not explain between-species differences in submergence tolerance. These species-wide responses to submergence could represent general responses to different signals that are also present in other environments, such as low photosynthate concentrations and high relative humidity.
We thank Hannie de Caluwe (Radboud University) for technical assistance, Heidrun Huber (Radboud University) for statistical advice and Hans de Kroon (Radboud University), Ronald Pierik and Thijs Pons (Utrecht University) for stimulating discussions.