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Keywords:

  • eutrophication;
  • Lobelia dortmanna;
  • organic enrichment;
  • oxygen;
  • anoxia;
  • photosynthesis;
  • sediment biogeochemistry;
  • stress

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Lobelia dortmanna thrives in oligotrophic, softwater lakes thanks to O2 and CO2 exchange across roots and uptake of sediment nutrients. We hypothesize that low gas permeability of leaves constrains Lobelia to pristine habitats because plants go anoxic in the dark if O2 vanishes from sediments.
  • We added organic matter to sediments and followed O2 dynamics in plants and sediments using microelectrodes. To investigate plant stress, nutrient content and photosynthetic capacity of leaves were measured.
  • Small additions of organic matter triggered O2 depletion and accumulation of NH4+, Fe2+ and CO2 in sediments. O2 in leaf lacunae fluctuated from above air saturation in the light to anoxia late in the dark in natural sediments, but organic enrichment prolonged anoxia because of higher O2 consumption and restricted uptake from the water. Leaf N and P dropped below minimum thresholds for cell function in enriched sediments and was accompanied by critically low chlorophyll and photosynthesis.
  • We propose that anoxic stress restricts ATP formation and constrains transfer of nutrients to leaves. Brief anoxia in sediments and leaf lacunae late at night is a recurring summer phenomenon in Lobelia populations, but increased input of organic matter prolongs anoxia and reduces survival.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Lobelia dortmanna L. inhabits sandy beaches of the most pristine oligotrophic, softwater lakes in Europe and North America (Sculthorpe, 1967). It maintains an evergreen biomass and displays the lowest metabolism and the slowest tissue turnover of any temperate aquatic plant (Moeller, 1978; Sand-Jensen & Søndergaard, 1978; Nielsen & Sand-Jensen, 1991). Lobelia has small, thick leaves in a rosette from a short stem and numerous roots with a higher surface area than the leaves (Sand-Jensen & Prahl, 1982). Large continuous air lacunae run through leaves and roots, a thick leaf cuticle reduces the permeability of O2 and CO2, while the roots have no barriers to gas exchange with the sediment pore water (Møller & Sand-Jensen, 2008). Thus, Lobelia has efficient intraplant transport of O2 and CO2 between leaves and roots and rapid gas exchange between roots and sediment. This plant–sediment coupling is enforced by extensive root symbiosis with arbuscular mycorrhiza fungi (Wigand et al., 1998). Although these features help Lobelia to acquire sediment CO2 and nutrients in nutrient-poor softwater lakes, they could also make the plant particularly susceptible to depletion of O2 and reduced conditions in sediments following higher sedimentation of labile organic matter (Sand-Jensen et al., 2005a; Raun et al., 2010). Our general objective was, therefore, to determine how O2 dynamics, sediment processes and Lobelia’s performance respond to enrichment of sediments with different amounts of labile organic matter.

Eutrophication of numerous Lobelia lakes has led to decline of Lobelia and other isoetid species (Sand-Jensen et al., 2000; Smolders et al., 2002; Geurts et al., 2008). The efficient intraplant transport of gases and the lack of diffusive barriers across the root surfaces mean that O2 is readily lost to the sediment. At high sediment O2 consumption, steep diffusive gradients result in large root O2 effluxes (Christensen et al., 1994; Sand-Jensen et al., 2005b) and problems in maintaining sufficient internal O2 transport along the roots to the tips (Armstrong et al., 2000; Møller & Sand-Jensen, 2008). Other aquatic plants often have diffusive root barriers to prevent radial O2 loss or produce barriers to maintain sufficient O2 transport to the root tips when sediments become reduced (Colmer, 2003). Lobelia does not form root tissue barriers because it requires CO2 for photosynthesis from the sediment (Møller & Sand-Jensen, 2008). As a result, roots of Lobelia and other isoetid species become shorter when grown in sediments with high oxygen demand (Sand-Jensen et al., 2005a; Raun et al., 2010), but shorter roots also have smaller root surfaces for absorption of mineral nutrients. Solute transport and mineral nutrition can be further compromised by O2 deprivation of roots and symbiotic root fungi because of cessation of ATP production by oxidative phosphorylation and thus a severe energy deficit (Colmer & Flowers, 2008). Anaerobic carbohydrate catabolism provides some ATP during anoxia, albeit only 3–35% of the rate of energy production in aerobic cells (Gibbs & Greenway, 2003; Greenway & Gibbs, 2003), but takes a higher toll on carbohydrates. Lobelia could be particularly sensitive to anoxia because of the observed low photosynthesis and carbohydrate production, high proportions by weight of nonphotosynthetic stem and root tissue and low capability of root tissue to speed up anaerobic fermentation and ATP production (Sand-Jensen & Søndergaard, 1979; Nielsen & Sand-Jensen, 1989; Smits et al., 1990).

O2 availability is the main determinant of sediment and plant processes. We therefore made a special effort to measure O2 continuously during light and dark periods in lake water, sediments and leaf lacunae to establish if, where and for how long hypoxia or anoxia occurred in laboratory and field populations subjected to gradually increasing organic enrichment and O2 consumption rates. Because Lobelia grows slowly and sediment processes respond slowly, we followed the responses for up to 194 d after organic enrichment to make sure that plant stress was fully expressed and recovery was possible.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Laboratory experiments with organic enrichment

Six intact sediment turfs were collected in mid-October from shallow water in a homogeneous Lobelia dortmanna population in oligotrophic Lake Värsjö, south-west Sweden. The low-organic sandy turfs (0.60 ± 0.05% (mean ± SD) organic matter of sediment DW) were 17 cm long and 15 cm wide and had a sediment depth of 12–14 cm that ensured intact root systems of c. 20 plants. Turfs were brought fully submerged to the laboratory for experiments. One turf was left as a control, while gradually longer rod-shaped dry pellets (5 mm in diameter, 1–15 mm long) of commercially available pasture grass (Dlg, Copenhagen, Denmark) were added to the other five turfs forming a gradient of added labile organic matter equivalent to 0 (control), 0.1, 0.2, 0.4, 0.8 and 1.6% organic matter of DW. Organic pellets contained (as a percentage of DW) 91 ± 1% (= 4) organic matter, 46.9 ± 0.4% organic carbon (C), 2.3 ± 0.15% total nitrogen (TN) and 0.27 ± 0.01% total phosphorus (TP). This composition is equivalent to weight proportions of 156 C, 8.5 N and 1.0 P, which is richer in nutrients relative to C and richer in P relative to N than Lobelia tissue (Sand-Jensen et al., 2005a). Pellets were inserted with pincers at 4 cm sediment depth with a fixed horizontal distance of 2 cm equivalent to 71 pellets per turf. Pincers were stuck into control sediments to keep physical disturbance constant. Previous experiments have documented that aerobic O2 consumption rates of surface sediments increase linearly with addition of this type of organic matter, generating an increasing O2 stress on plant roots (Raun et al., 2010). The turf specimens were incubated at 14–16°C in 80 l aquaria with a 12 : 12 h light : dark cycle at an irradiance of 110 μmol photons m−2 s−1 (photosynthetically active radiation, PAR) and exposed to the same water to eliminate possible effects of elevated carbon and nutrient availability in the water resulting from release from enriched sediments. The water was renewed every second week to keep nutrient concentrations low and avoid algal growth and it atmospheric air was bubbled slowly through it to ensure mixing and air saturation. Because large water volumes were needed we used filtered water from nearby Lake Esrum diluted 20 times with demineralized water to obtain a chemical composition closely resembling that of Lake Värsjö. After dilution mean values were: 0.13 mM inorganic carbon (DIC), 0.3 μM plant available P (ortho-P), 0.4 μM NO3 and 0.3 μM NH4+.

Pore-water chemistry and sediment characteristics

Repeated pore-water samples from 1, 2, 4, 6 and 8 cm depth were extracted from three representative sites distributed evenly across the turf area at the end of the 194-d-long experiment and analysed for dissolved Fe2+, DIC, pH, NH4+ and ortho-P. Pore-water was sampled by inserting thin capillary glass tubes (1 mm) in the sediment, keeping the upper end above the water. Capillary forces and pressure difference slowly and steadily filled the capillary tube with water from the desired sediment depth within a few minutes. Quantities of 50–100 μl of pore-water were withdrawn with a glass syringe from each of five capillary tubes at a certain sediment site to yield an integrated sample for 0–8 cm depth with minimum air contact and these were analysed immediately. Reduced Fe2+ was measured spectrophotometrically according to the phenanthrolin method (Eaton et al., 1995). DIC was determined by injecting a minute pore-water volume into 3% HNO3 in a bubble chamber purged with N2 gas carrying evolved CO2 into an Infrared gas analyzer (IRGA, ADC-225-MK3, Hoddesdon, UK) as previously described (Vermaat & Sand-Jensen, 1987). pH was measured by a flat-membrane pH electrode (LoT403-M8-S7/120, Mettler Toledo, Greifensee, Switzerland) positioned close to a glass surface allowing pH measurements on small droplets injected between the electrode membrane and the glass surface. Free CO2 was calculated from DIC, pH, ionic strength and temperature (Rebsdorf, 1972). NH4+ was measured spectrophotometrically on 100 μl pore water by diluting with 900 μl distilled water and adding 100 μl phenol and 100 μl hypochlorite reagents according to a microversion of Solórzano (1969). Analysis of dissolved ortho-P according to Strickland & Parsons (1968) initially caused analytical problems as a result of very low P concentrations and high blanks resulting from nonremovable colloidal organic matter. Measurements succeeded when pore water from each sediment and depth without reagents served as blanks for parallel samples with reagents added.

After the experiment, sediment composition was characterized in duplicate sediment cores by analysing wet and dry weight in samples from depth intervals of 0–1 cm, 1–3 cm, 3–5 cm and 5–8 cm. Dried homogenized samples were then analysed for TN and organic carbon (C) on a CHN EA1108-elemental analyser (Carlo Erba Instruments, Milan, Italy), TP by the method of Andersen (1976), total iron (TFe) by the phenanthrolin method slightly modified by Møller & Sand-Jensen (2008) and organic content as loss on ignition at 550°C.

Plant morphology and performance

Plant morphology and metabolism were measured on one plant of each treatment 18, 59 and 109 d into the experiment, while three plants were killed after 194 d. In general, plants within the same treatment looked very similar. Therefore, representative plants could easily be selected. Plants were gently removed from the sediment to minimize disturbance. Total leaf and root lengths were measured on each plant by a ruler. Two replicates for measurements of photosynthesis and respiration were prepared from each harvested plant. Two leaves for each replicate were split longitudinally through the lacunae to facilitate CO2 supply and avoid the influence of O2 storage in the extensive lacunae and then transferred to gas-tight glass bottles containing alkaline water adjusted to pH 6.9 to provide a high saturating CO2 concentration of 1000 μM for photosynthesis. The bottles were transferred to a rotating wheel in an incubator at 16°C and exposed to a saturating irradiance of 340 μmol photons m−2 s−1 (PAR). Net photosynthesis was calculated as the increase in O2 concentration after 2 h in the light. O2 concentration was measured with a Clark type OX500 mini-electrode (Unisense, Århus, Denmark). Photosynthesis was normalized to leaf DW determined after 24 h of freeze-drying. Photosynthetic capacity measured under these standardized experimental conditions is suitable for evaluating the physiological well-being of leaves, but does not directly reflect photosynthesis in the growth experiments because of different supplies of sediment CO2 to Lobelia between treatments.

Chlorophyll was measured on leaves used for photosynthetic experiments by ethanol extraction for 24 h and spectrophotometric analysis according to Christoffersen & Jespersen (1986). TN, TP and TFe were measured on leaves from three to five different plants at different times during the experiments and analysed by methods already described for sediments.

O2 dynamics and penetration depth

O2 dynamics in plants and sediment were measured on four occasions in different plants during the laboratory experiment using Clark-type O2 micro- and mini-electrodes (Ox 50, tip diameter 50 μm for leaf lacunae and Ox 500, sturdy type applicable for sediments with a tip diameter of 500 μm) advanced by micromanipulators (Unisense). O2 was recorded using a picoamperometer connected to a computer enabling continuous logging of signals. Likewise, temperature was measured continuously using thermocouples (type K) connected to the computer through a DC10 converter and used to correct the electrode signal for temperature changes. Electrodes were inserted into lacunae of mature leaves number 3–4 in the rosette and into sediments at the desired depth and left for a full 12 : 12 h light : dark cycle. Electrodes were calibrated before and after experiments in water bubbled with atmospheric air and in O2-free water obtained by adding dithionite. According to the manufacturer, the detection limit of microelectrodes for O2 is 0.03 kPa. However, because of temperature variations in our measurements, determinations under confirmed anoxic conditions suggest that the operational detection limit is 0.1 kPa. Nonetheless, we still use the term anoxia for sediments and leaf lacunae in the dark below 0.1 kPa, because O2 concentrations declined steeply before reaching the steady minimum O2 signal, stressing that O2 consumption rates exceeded O2 supply rates to such a great extent that it is unlikely that hypoxia characterized by trace amounts of O2 (i.e. < 0.1 kPa) existed.

O2 penetration depth was measured in duplicate in each sediment turf on nine occasions during the course of experiments, and in triplicate at termination with the same equipment used for measuring sediment O2 dynamics. Measurements were always made 10–12 h into the light period when O2 penetrated deepest. The O2 electrode was moved downwards in the sediment by a micromanipulator in steps of 500 μm until anoxia occurred or the depth range of the manipulator prevented deeper measurements (> 40 mm).

Field experiments with organic enrichment

Field experiments were performed in summer–autumn in the same shallow homogeneous Lobelia population in Lake Värsjö as used for collection of sediment turf for laboratory experiments to make sure that patterns observed in laboratory experiments resembled natural sediment and plant responses in the field.

A long-term organic enrichment experiment was made over 80 d in the field (10 July–1 October) in which sediment chemistry and plant performance were compared between triplicate controls and treatments (20 × 20 cm per plot) enriched with organic matter to 0.8% DW as described for the laboratory experiment. At the end of the experiment, pore-water concentrations and sediment composition were measured on sediment cores (5 cm in diameter, 10 cm in depth) taken from each plot and brought back to the laboratory in Perspex tubes with rubber stoppers preventing disturbance of sediment and pore water. Cores were stored in the laboratory at 15°C in a 12 : 12 h light : dark cycle for 1–4 d before pore water was extracted and analysed as already described. Likewise, net photosynthesis was measured in the laboratory incubator on leaf samples from each plot. Leaf samples derived from harvested field plants were transported to the laboratory in sealed moist plastic bags and stored at 8°C and used for experiments within 2 d.

O2 dynamics was measured continuously over a full 24 h light–dark cycle in the field in late August in duplicated experiments by inserting microelectrodes into leaf lacunae of two plants and inserting mini-electrodes at 1 cm depth in the sediment next to these two plants using the same type of equipment as for laboratory experiments. HOBO data loggers were used to measure water temperature and irradiance (Onset Computer Corporation, Bourne, Massachusetts, USA).

Data treatment and statistical analysis

Data were processed in Excel 2007 and statistical analyses and graphs were made in Graph Pad Prism 5. Data are presented as means ± SD. < 0.05 was considered significant. The in situ experiment was a block design in triplicate suitable for t-test analysis, whereas the laboratory experiment was a gradient study with six amounts of added organic matter to single sediment turfs inhabited by 20 plants (pseudo-replicates) suited for regression/correlation analysis. Values from the laboratory experiment are presented as means ± SD of usually three replicates from each sediment turf.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Sediment processes and chemistry

Organic enrichment of sediments in long-term laboratory experiments stimulated O2 use for degradation of organic matter and reduced O2 depth penetration (Fig. 1). O2 penetration depth exceeded 40 mm in control sediments but declined to < 3 mm after 25 d in sediments fertilized with ≥ 0.4% organic matter. In sediments receiving only 0.1 and 0.2% organic matter, O2 penetration depth had only declined to 21 and 10 mm, respectively, 25 d after fertilization. As degradation of organic matter progressed over time, O2 gradually penetrated deeper into the sediments. However, only at the lowest organic dose did O2 penetration recover to the same value (> 40 mm) as in the control sediment within the 195 d of experiments. At the end of the experiment, O2 penetration depth was significantly and negatively correlated to the magnitude of addition (Spearman’s r, < 0.001). Thus, organic enrichment had profound and long-lasting effects on O2 availability and decomposition processes.

image

Figure 1. Depth of O2 penetration in Lobelia sediments as a function of time after addition of different amounts of labile organic matter (per sediment DW) (open circles, control; closed circles, 0.1%; open squares, 0.2%; closed squares, 0.4%; open diamonds, 0.8%; closed diamonds, 1.6%). Measurements were made 10–12 h into the 12 h light period. Measurements could not extend deeper than 40 mm into the sediment, when O2 penetration in control sediments was described as > 40 mm. Values are mean of two (0–170 d) and three (194 d) measurements ± SD.

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This persistent enrichment effect was evident in sediment chemistry even at the lowest enrichment after 194 d in the laboratory experiment (Table 1). Across the gradient, organic content, TN and TP increased two- to threefold and they were significantly correlated to the amount of organic matter added (linear regression, < 0.0001), while water content and TFe did not change significantly. Approximately 14.5 mg organic matter, 368 μg TN and 43 μg TP (all g–1 dry sediment) were added with the highest 1.6% organic enrichment and the treated sediment still contained an extra 10.2 mg organic matter, 382 μg TN and 25.5 μg TP relative to the control sediment after 194 d. Likewise, the 0.4% organic treatment received an additional 3.6 mg organic matter, 92 μg TN and 9.8 μg TP per g dry sediment and still contained extra 2.3 mg organic matter, 96 μg TN and 9.8 μg TP after 194 d, stressing that organic matter was lost by degradation but most N and P remained in the sediment.

Table 1.   Organic content (%), water content (%), total nitrogen (TN), total phosphorus (TP), total iron (TFe) all normalized to sediment DW and mean depth-integrated concentrations of Fe2+, NH4+, dissolved inorganic carbon (DIC) and CO2 measured in sediments after 195 d of experimentation in the laboratory across a gradient of added labile organic matter (0–1.6% DW)
TreatmentSediment compositionPore-water concentration
Organic matter added (%)Organic content (%)Water content (%)TN (μg g−1)TP (μg g−1)TFe (mg g−1)Fe2+ (μM)NH4+ (μM)CO2 (mM)DIC (mM)
  1. r2 and significance levels of linear regressions between the dependent variable and % organic matter added are shown.

  2. Mean ± SD, = 2 (sediment composition) or = 3 (pore water). ns, not significant.

00.60 ± 0.0531.1 ± 0.5170 ± 2024.1 ± 2.31.97 ± 0.130003 ± 34.1 ± 0.80.52 ± 0.091.99 ± 0.15
0.10.68 ± 0.0229.7 ± 0.3186 ± 1027.6 ± 0.31.91 ± 0.010070 ± 4422.6 ± 110.67 ± 0.022.34 ± 0.27
0.20.66 ± 0.0528.7 ± 0.3193 ± 2029.1 ± 1.81.93 ± 0.000510 ± 117200 ± 131.03 ± 0.034.53 ± 0.12
0.40.83 ± 0.1631.1 ± 0.4266 ± 1433.9 ± 2.21.87 ± 0.040979 ± 13590 ± 761.78 ± 0.067.01 ± 0.02
0.81.06 ± 0.1630.7 ± 1.0294 ± 8.944.0 ± 2.12.02 ± 0.061989 ± 107850 ± 952.18 ± 0.097.89 ± 0.57
1.61.62 ± 0.3730.9 ± 1.7552 ± 24149.6 ± 3.31.79 ± 0.142700 ± 152568 ± 2034.14 ± 0.177.72 ± 0.50
Correlationr= 0.96, < 0.0001r= 0.03, nsr= 0.80, < 0.0001r= 0.90, < 0.0001r= 0.11, nsr= 0.94, < 0.0001r= 0.45, < 0.001r= 0.96, < 0.0001r= 0.61, < 0.0001

In sediment pore water examined at the end of experiments, DIC and CO2 increased four- to eightfold and NH4+-N increased > 100-fold with the 1.6% organic enrichment reflecting the enhanced organic decomposition (Table 1). A 1000-fold increase of soluble Fe2+ is the result of use of Fe3+ by microorganisms (i.e. use of alternative electron acceptors) and it is one of the main reasons for the increase of HCO3 contained in DIC (Lucassen et al., 2009). Dissolved ortho-P concentrations significantly different from zero could not be detected in sediments with 0–0.8% added organic matter, but in the 1.6% organic treatment high concentrations were recorded (i.e. 328 ± 176 μg P g−1 sediment DW at 4 cm depth).

In field experiments, pore-water chemistry responded as in laboratory experiments. Significantly higher pore-water concentrations of DIC (4.8 ± 0.95 mM) and Fe2+ (1.03 ± 0.54 mM) were found in 0.8% organic treatments after 80 d compared with 1.0 mM DIC and 0.02 mM Fe2+ in control sediments (t-test, < 0.01), reflecting stimulation of organic decomposition, sediment anoxia and alkalinization by Fe reduction (Table 2).

Table 2.   Maximum net photosynthesis (NP), chlorophyll (chl), total phosphorus (TP) and total nitrogen (TN) content of Lobelia leaves in relation to DW, sediment organic content and pore-water concentrations of Fe2+ and dissolved inorganic carbon (DIC) at 4 cm depth after 80 d of experiments with sandy sediments receiving 0 and 0.8% labile organic matter in Lake Värsjö from 10 July to 1 October
TreatmentPhotosynthesisLeaf contentSediment compositionPore water
Organic matter added (%)NP (μmol O2 g−1 DW h−1)Chl (mg g−1 DW)TP (mg g−1 DW)TN (mg g−1 DW)Organic content (%)Fe2+ (μM)DIC (mM)
  1. Mean ± SD, = 3.

0.0282 ± 182.47 ± 0.341.62 ± 0.2623.2 ± 4.320.66 ± 0.1016.0 ± 5.611.03 ± 0.54
0.864 ± 170.99 ± 0.150.89 ± 0.0211.3 ± 2.210.81 ± 0.031204 ± 3814.78 ± 0.95

O2 dynamics in plant lacunae and sediments

In leaf lacunae of Lobelia on nonenriched sediments in laboratory experiments, O2 changed from 27–33 kPa (130–160% saturation) late in the light period to anoxia late in the dark period (Fig. 2a). Diurnal O2 changes in sediment pore water at 10 mm depth tracked these changes very closely with a 1 h time lag upon changes to light or darkness (Fig. 2a). By contrast, leaf lacunae rapidly became anoxic in the dark in Lobelia grown for 9 or 7 d on sediments enriched with 0.4 (Fig. 2b) and 1.6% organic matter, respectively (Fig. 2c). Pore water at 10 mm depth was already permanently anoxic 9 d after enrichment with 0.4% organic matter. The 1.6% organic treatment still contained some O2 in the sediment pore water in the light 7 d after organic enrichment because O2 production by photosynthesis was initially very high according to the steep rise of O2 in the leaf lacunae upon illumination. Photosynthesis is probably stimulated by high sediment CO2 accompanying faster organic decomposition (Fig. 2c). However, in all later incubations, sediments enriched with 0.4 and 1.6% organic matter remained permanently anoxic at 10 mm depth, whereas the control treatment had similar sediment O2 traces as first observed (data not shown).

image

Figure 2. Diurnal changes of O2 partial pressure (PO2) in leaf lacunae of Lobelia (solid line), sediment pore water at 10 mm depth (dashed line) and water phase (dotted line) in laboratory experiments in the control (a), 0.4% (b) and 1.6% (c) treatments after 4, 9 and 7 d of enrichment, respectively. The diurnal trace started with a shift from 12 h light to 12 h darkness followed by a shift back to 12 h light. Values are single measurements.

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During the 194-d-long laboratory experiment, the diurnal fluctuation of O2 in the leaf lacunae remained almost the same in Lobelia growing on unfertilized sediments (Fig. 3). O2 increased to c. 30 kPa during daytime photosynthesis and declined to anoxia late at night. Depending on the specific experiment, maximum daytime O2 concentrations in leaf lacunae of Lobelia on sediments enriched with 0.4% organic matter ranged from 15 to 43 kPa and they rapidly went anoxic and stayed anoxic for several hours in the dark. Daytime O2 in leaf lacunae of Lobelia on sediments enriched with 1.6% organic matter never exceeded 20 kPa after 40 d into the experiment and O2 disappeared more rapidly in the dark than in the 0.4% organic treatment.

image

Figure 3. Diurnal changes in O2 partial pressure (PO2) in leaf lacunae of Lobelia with increasing time into the experiment (a, 4–9 d; b, c. 40 d; c, c. 73 d; d, c. 150 d (control and 1.6%) and 111 d (0.4%)) for plants growing in the laboratory in sediments with 0% (control, solid line), 0.4% (dashed line) and 1.6% addition (dotted line) of labile organic matter per sediment DW. Data in (a) are derived from Fig. 2. The diurnal trace started with a shift from 12 h light to 12 h darkness followed by a shift back to 12 h light. Different plants from each treatment were used for measurements over time, = 1.

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The duration of leaf anoxia during the 12 h dark period was significantly positively correlated to % organic matter added (Spearman’s r, < 0.01, joint analysis of all measurements; Fig. 4). As root morphology changed and degradation of organic matter progressed towards the end of the experiment, the anoxic period in leaf lacunae in the dark declined to 1.8 h on control sediments and to 5.9 and 6.6 h on sediments enriched with 0.4 and 1.6% organic matter, respectively (Fig. 4). The decline of the anoxic period with time of the experiment was systematic in all treatments, although not statistically significant.

image

Figure 4. Duration of anoxia during the 12 h dark period in leaf lacunae of Lobelia as a function of time after addition of different amounts of labile organic matter (circles, control; squares, 0.4%; diamonds, 1.6% of added organic matter to sediments). Different plants from each treatment were used for measurements over time (n = 1).

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Field experiments with Lobelia populations in late August under the same temperatures, a 13 : 11 h light : dark cycle and higher daytime irradiance displayed the same diurnal O2 course in leaf lacunae and sediment pore water at 1 cm depth as in laboratory experiments (Fig. 5). Thus, O2 in the sediment reached 20–23 kPa (close to 100% saturation) late in the afternoon and vanished for 1–6 h late in the night. In the leaf lacunae, O2 peaked at 22–31 kPa late in the afternoon and vanished for 1–3 h late in the night.

image

Figure 5. Diurnal changes of O2 partial pressure measured at 10 mm depth in sandy sediments next to two different plants (a, solid line), in leaf lacunae of two plants (b, solid line) and in the water phase (a and b, dotted lines) in Lobelia populations in Lake Värsjö in late August. Irradiance (photosynthetically active radiation, 400–700 nm; c, solid line) and water temperature (c, dashed line) are also shown.

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Chlorophyll content and photosynthesis

Chlorophyll content and photosynthesis at saturating light and CO2 changed in concert across organic enrichments and over time in laboratory experiments (Fig. 6). Chlorophyll content and maximum photosynthesis were highest and at approximately the same level among organic enrichments 18 d into the experiment. A small increase of both chlorophyll content and photosynthesis sometimes occurred at low organic enrichment (0.1 and 0.2%); however, when omitting the control treatment from the dataset, both variables subsequently declined significantly (linear regression, < 0.05) across the range of organic enrichments. Photosynthesis dropped to very low amounts in the 1.6% organic enrichment.

image

Figure 6. Chlorophyll content (a) and maximum net photosynthesis (b) of Lobelia leaves after increasing addition of labile organic matter (% of sediment DW). Single measurements were made after 18 d (open circles) and 59 d (closed circles) and triplicate measurements (± SD) after 194 d (squares) of experiments in the laboratory.

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For all leaf samples among treatments and over time, chlorophyll content was significantly positively related to tissue concentrations of TN and almost so to TP (linear regression, P = 0.06, Table 3). Chlorophyll was significantly negatively related to TFe because some leaves from the 1.6% organic treatment had surface plaques of Fe. TP and TN were also significantly interrelated. Photosynthesis was highly significantly related to chlorophyll, TP and TN in the leaves, while it was significantly negatively related to TFe (Table 3). Also in field experiments, photosynthesis, chlorophyll, TN and TP declined significantly with 0.8% organic enrichment (t-test, < 0.01; Table 2).

Table 3.   Summary of linear regressions between dependent variable (y) and independent variable (x)
yxSlopeInterceptr2Pn
  1. Ratio of leaf to root length (L : R) to sediment enrichment (org, % DW) after 195 d and relationship between chlorophyll content (Chl, mg g−1 DW), net photosynthesis (NP, μmol O2 g−1 DW h−1), TN content (mg g−1 DW), TP content (mg g−1 DW) and TFe content (mg g−1 DW) of Lobelia leaves retrieved after different experimental periods (18–194 d) in the laboratory from sediments of different organic enrichment. Slopes, intercepts, significance levels (P), r2-values, and number of data points (n) used for calculations are presented [correction added after online publication 27 January 2011: some values in the columns headed ‘Slope’ and ‘Intercept’ have been altered from negative values to positive values by the removal of minus signs; additionally within the Probability ‘(P)’ column, some significance levels have been altered by the removal of less than (<) signs].

L : ROrg 0.78−0.640.83< 0.000118
ChlTFe−3.841.030.510.000123
ChlTP 0.76−2.630.160.059523
ChlTN 0.11−6.140.340.002624
TNTP 7.68−1.310.68< 0.000123
NPChl 43.10.010.59< 0.000172
NPTFe−1811.010.270.010523
NPTP 90.4−0.370.55< 0.000123
NPTN 8.803.570.51< 0.000124

Root development and leaf nutrients

Maximum root length declined from c. 7.1 ± 1.6 cm in control sediments to only 4.3 ± 0.5 cm in the organically richest sediments deprived of O2 in laboratory experiments. Across the organic enrichment gradient, the ratio of leaf length to root length increased fourfold from 0.53 ± 0.1 in the control to 1.89 ± 0.1 in the 1.6% organic matter treatment (Table 3).

Total phosphorus and TN in leaf tissue changed profoundly among treatments and over time in both laboratory and field experiments (Fig. 7, Table 2). The highest and most constant leaf concentrations occurred in plants from control sediments, but progressively lower TP and TN concentrations occurred with duration of the experiment and higher addition of organic matter despite increasing nutrient concentrations in the sediments (Tables 1, 2). Tissue TP remained close to the common critical threshold for maximum biomass yield of submerged macrophytes in control sediments and in sediments enriched with only 0.1 and 0.2% organic matter (Fig. 7, Table 2). However, depletion of TP was more extreme and concentrations dropped below common critical threshold for maximum growth rate, maximum biomass yield and even minimum threshold to sustain growth of submerged macrophytes at 0.4–1.6% organic enrichment (Fig. 7, Table 2). Depletion of TN in leaf tissue occurred later during the experiment and at higher additions of organic matter, and TN concentrations only dropped below the critical threshold for maximum biomass yield at the highest organic treatment towards the end of experiments.

image

Figure 7. Total phosphorus (TP) (a) and total nitrogen (TN) (b) concentrations in Lobelia leaves at different days (open circles, 18 d; closed circles, 56 d; open squares, 120 d; closed squares, 194 d) after enrichment of sediments with 0–1.6% labile organic matter in the laboratory. Values are single measurements after 18, 56 and 120 d and mean (± SD) of triplicates after 194 d. Mean critical TP and TN thresholds in leaves suggested to limit maximum growth rate (arrow A), maximum biomass yield (arrow B) and stop all development (arrow C) of aquatic vascular macrophytes are derived from Gerloff & Krombholz (1966), Hutchinson (1975), Colman et al. (1987) and Demars & Edwards (2007). Critical nutrient concentrations could be lower in Lobelia than the mean values for many aquatic macrophytes.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

O2 dynamics in sediments and plants

Laboratory experiments reflected the natural processes in the field. We observed the same profound O2 fluctuations from above air saturation in the light to anoxia in the dark in the sandy sediments and also the same plant responses in the two instances. It was surprising, however, that leaf lacunae tracked O2 conditions in the sediment pore water so closely and that they went anoxic in the dark shortly after O2 had disappeared from the sediment. In a November experiment at 4°C in the same Lobelia population, O2 remained in leaf lacunae (9 kPa), root lacunae (6 kPa) and in the sediment (3 kPa) in the dark because plant and sediment respiration is reduced at low winter temperature (Sand-Jensen et al., 2005b). Leaf and root anoxia is, however, a recurring phenomenon during summer nights in field populations of Lobelia despite 100% air saturation in lake water and large retention capacity for O2 in the extensive air lacunae (Sand-Jensen & Prahl, 1982). The impeded gas exchange across leaf surfaces as a result of very low permeability (Sand-Jensen & Prahl, 1982; Pedersen & Sand-Jensen, 1992) implies that air lacunae and plant tissue go anoxic only 1–2 h after O2 has disappeared from the sediment. Lobelia has to tolerate this stress every night, reduce metabolism and use fermentation to cover energy demands for 2–4 h. This daily event does not threaten plant performance and survival as long as the anoxic period is short. However, even moderate addition of labile organic matter (0.1–0.2%) reduced sediment O2 profoundly, and higher organic doses (0.4–1.6%) prolonged anoxia in the dark to 7–9 h with accompanying detrimental consequences for plant nutrition, root growth and photosynthesis. A fourfold decline of photosynthesis was also observed in field populations enriched with 0.8% organic matter for 80 d (Table 2).

L. dortmanna is one of the very few species that can occupy wave-exposed beaches with nutrient-poor mineral sediments and low CO2 availability because of its outstanding stress-selected traits (Farmer & Spence, 1986).These traits comprise strong root anchorage of a flat leaf rosette and low rates of metabolism, growth and mortality (Sand-Jensen & Søndergaard, 1978). Photosynthesis is typically 10-fold lower for Lobelia than for most nonisoetid species (Nielsen & Sand-Jensen, 1989) and Lobelia leaves remain alive for 1–2 yr compared with only 1–2 months for most nonisoetid species (Sand-Jensen & Borum, 1991). Lobelia is particularly adapted to its environmental conditions, as it stimulates aerobic sediment degradation by root O2 release, acquires P through symbiotic fungi and maintains CO2 for use in photosynthesis within the sediment–plant association because of impermeable leaves (Pedersen et al., 1995; Wigand et al., 1998). However, Lobelia is also confined to chronically oligotrophic habitats because it cannot tolerate even moderately high O2 consumption rates of the sediments as demonstrated here, and it lacks adaptations to cope with shading from dense growth of phytoplankton, epiphytic algae and tall-rooted plants (Sand-Jensen et al., 2000; Geurts et al., 2008). In the face of global warming stimulating benthic plant growth and sediment O2 consumption, we may predict that Lobelia’s distribution will be further constrained and that its southern distribution limit will be pushed to the north.

We cannot interpret the particular anatomy, morphology and performance of Lobelia, resulting in virtually all O2 involved in photosynthesis and respiration being exchanged via the roots, as an adaptation to attain advantageous O2 dynamics. On the contrary, anoxia in the plant in the dark as a result of the strong gas diffusion barrier to leaf uptake represents a high potential risk of stress even in its natural oligotrophic habitat. Lobelia’s special structure can instead be interpreted as an adaptation to use CO2 in the sediment pore water as a much richer carbon source for photosynthesis than the surrounding water. Pore water in natural sandy sediments contained 0.60 mM CO2 (Table 1), which is 40 times higher than CO2 concentrations in air-saturated lake water (c. 0.015 mM). Lobelia can only use CO2 for photosynthesis and air-saturated concentrations are too low to support positive net photosynthesis (Winkel & Borum, 2009). To ensure high CO2 supply from the sediment to leaf photosynthesis, root surfaces must be large and highly permeable and air lacunae must be wide and short through roots and leaves. Moreover, to maintain high CO2 concentrations in plants growing on sediments of low decomposition rates, plants must prevent CO2 loss to the water via the intraplant gas transport route and this requires a lid (a diffusion barrier) on leaf surfaces.

The diffusion barrier on Lobelia’s leaves also reduces evaporation and ensures survival when plants regularly become exposed to the air following drawdown of the water table during summer (Pedersen & Sand-Jensen, 1992). Most submerged aquatic plants dry out upon exposure to air and several species, including the common isoetid Littorella uniflora (Nielsen et al., 1991), survive by producing new aerial leaves with stomata. Lobelia does not invest in a new set of leaves, which would be costly and perhaps impossible considering its low intrinsic growth rate and nutrient-poor habitat. Thus, special structural and physiological adaptations serve several purposes and need to be viewed in regard to the entire plant life.

Environmental changes and plant stress

Sediment chemistry was very susceptible to addition of modest amounts of easily degradable organic matter. O2 disappeared from most of the sediment in the light with addition of only 0.1 or 0.2% organic matter and it took 100 d for O2 to resume penetration deeper than 40 mm at the lowest dose. Even after 194 d, pore-water concentrations of DIC, CO2, NH4+ and Fe2+ were elevated relative to control values, reaching the substantial 200 μM NH4+ and 510 μM Fe2+ at 0.2% organic addition (Table 1). These low organic treatments did not reduce leaf nutrients, chlorophyll and photosynthesis, although such high NH4+ concentrations stress other macrophytes when leaves are directly exposed (Smolders et al., 1996).

Higher additions of organic matter (0.4–1.6%) clearly impeded photosynthesis and incorporation of TP and TN in the leaves. The most parsimonious explanation for the stress is prolonged anoxia in leaves and roots in the dark reducing ATP production by oxidative phosphorylation to sustain uptake, transport and incorporation of mineral ions and organic solutes in cell products (Aguilar et al., 2003; van Dongen et al., 2003). A crucial role of TP and TN in leaf tissue for forming the photosynthetic apparatus is supported by significant positive correlations between TP, TN, chlorophyll and photosynthesis. According to high NH4+ concentrations in all organic amendments and high ortho-P concentration in the 1.6% treatment, it is not a lack of N and P in the sediment that restricts leaf nutrients, but insufficient uptake from the sediment and transfer to leaf tissue.

Anoxia initiates the accumulation of NH4+ and the formation of Fe2+ and other reduced compounds in the sediment (e.g. Mn2+, sulphides and small fatty acids), which may contribute to plant stress (Gibbs & Greenway, 2003). These compounds accumulate as a consequence of anoxia and do not have the same fundamental physiological influence as many hours of anoxia in the plant tissue. Also, insufficient ATP formation and gradual depletion of carbohydrate reserves can account for the inability to incorporate N and P in leaf tissue despite high sediment availability. Moreover, high concentrations of NH4+ and Fe2+ without any apparent stress on photosynthesis developed in sediment with 0.2% organic addition, i.e., concentrations only two to five times lower than observed at greater organic amendments. This result supports the notion that O2 deprivation is the overriding stress factor.

Phosphorus in leaf tissue declined below the general minimum threshold to support photosynthesis and growth of submerged macrophytes at organic amendments of 0.4–1.6% (legend to Fig. 7). Although the critical threshold is an average for many macrophytes and Lobelia may have lower requirements than most other species (Moeller, 1978; Demars & Edwards, 2007), leaf P, chlorophyll and photosynthesis are so low in the 1.6% organic treatment that Lobelia can just barely survive. These results imply that P limitation in Lobelia represents a strong additional stress of organic enrichment and O2 deprivation as a result of one or more mechanisms. First, inorganic P remains adsorbed to soil particles or gradually thicker Fe-coatings formed on root surfaces in reduced sediments (Christensen & Sand-Jensen, 1998). Second, and probably most significantly, reduced root uptake, translocation and leaf incorporation of P are the result of O2 stress and insufficient ATP production (Gibbs & Greenway, 2003). Thirdly, widespread anoxia is proposed to reduce uptake and translocation of P by mycorrhiza fungi (Wigand et al., 1998). TN depletion in leaf tissue is less severe, perhaps because of continued diffusive uptake of NH4+ from high pore-water concentrations (Marschner, 1995), which does not require active root membrane uptake or transfer by fungi.

In summary, anoxia in sediments and leaf lacunae late at night was a surprising recurring summer phenomenon in pristine Lobelia populations on nutrient-poor sandy sediments. Small additions of labile organic matter drastically reduced O2 depth penetration and prolonged leaf anoxia because impermeable leaf surfaces prevented O2 supply from the lake water. Prolonged anoxia and insufficient ATP production to sustain root uptake and translocation can account for the decline of TP below critical thresholds to sustain optimum photosynthesis, growth and cell function. Elevated NH4+, Fe2+ and other reduced compounds in enriched sediments can further enhance plant stress, but prolonged anoxia alone can account for the observed poor performance of Lobelia.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank The Willum Kann Foundation for financial support to this study through The Centre of Excellence for Research on lake Restoration (CLEAR). We thank Birgit Kjøller and Ole Pedersen for technical assistance and three anonymous referees for constructive comments.

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  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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