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Shoot atmospheric contact is of little importance to aeration of deeper portions of the wetland plant Meionectes brownii; submerged organs mainly acquire O2 from the water column or produce it endogenously in underwater photosynthesis
SARAH MEGHAN RICH,
School of Plant Biology (M084), Faculty of Natural and Agricultural Sciences
Future Farm Industries CRC, The University of Western Australia (M081), 35 Stirling Highway, Crawley, WA 6009, Australia
Partial shoot submergence is considered less stressful than complete submergence of plants, as aerial contact allows gas exchange with the atmosphere. In situ microelectrode studies of the wetland plant Meionectes brownii showed that O2 dynamics in the submerged stems and aquatic roots of partially submerged plants were similar to those of completely submerged plants, with internal O2 concentrations in both organs dropping to less than 5 kPa by dawn regardless of submergence level. The anatomy at the nodes and the relationship between tissue porosity and rates of O2 diffusion through stems were studied. Stem internodes contained aerenchyma and had mean gas space area of 17.7% per cross section, whereas nodes had 8.2%, but nodal porosity was highly variable, some nodes had very low porosity or were completely occluded (ca. 23% of nodes sampled). The cumulative effect of these low porosity nodes would have impeded internal O2 movement down stems. Therefore, regardless of the presence of an aerial connection, the deeper portions of submerged organs sourced most of their O2 via inwards diffusion from the water column during the night, and endogenous production in underwater photosynthesis during the daytime.
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Soil waterlogging and standing water can present significant stresses to most terrestrial plant species. Roots in inundated, anoxic soils are reliant on internal O2 movement from the shoots. Complete submergence of shoots typically compounds the considerable stress of sediment waterlogging as direct access to atmospheric CO2 and O2 is cut off. High resistance across leaf cuticles and boundary layers in conjunction with slow gas diffusion rates in water limit gas uptake and inhibit photosynthesis when under water (Smith & Walker 1980; Mommer et al. 2005; Colmer, Winkel & Pedersen 2011). Partial shoot submergence is considered to be less stressful as the aerial connection allows for exchange of gases with the atmosphere and movement within wetland plants from shoots to roots via low-resistance gas pathways (aerenchyma) (Bailey-Serres & Voesenek 2008; Colmer & Voesenek 2009).
Extensive stem aerenchyma is a common feature of aquatic plants (Sculthorpe 1967; Jung, Lee & Choi 2008) and is also often developed within wetland species as a response to inundation (Jackson & Armstrong 1999). Within the stems of aquatic plants, aerenchyma can develop in many patterns, with honeycomb (numerous scattered small lacunae), wheel (large ovate lacunae separated by ‘spokes’) and hollow (large single pith cavity) being the most common types (Jung et al. 2008). These same patterns are found in the stems of emergent wetland species; however, there is scant information on the formation and occurrence of stem aerenchyma in these plants in response to flooding.
Field studies of plant O2 dynamics have focused on completely submerged plants, mainly aquatic and marine plants which typically grow submerged (Greve et al. 2003; Borum et al. 2005, 2006; Sand-Jensen et al. 2005; Holmer et al. 2009), although there is one study of a submerged terrestrial plant with succulent, non-aerenchymatous shoots (Pedersen, Vos & Colmer 2006). Studies of stem O2 dynamics in partially submerged plants are limited, although deepwater rice, which has stems containing a large pith (medullary) cavity and some degree of cortical aerenchyma, has received attention. Setter et al. (1987) found no relationship between the floodwater O2 concentrations and the internodal lacunae gas concentrations of deepwater rice in the field with shoot tips emergent and showed internodal O2 concentrations to be below atmospheric levels (50% of atmospheric at a depth of 0.8 m). However, in a controlled flooding experiment also of deepwater rice with shoot tips emergent, Stünzi & Kende (1989) found that although internodal gas concentrations were lower at night, these recovered to near atmospheric levels during the day, even at a depth of 0.9 m. Concurrent studies of the O2 dynamics of partially and completely submerged wetland plants in a field situation are lacking.
This paper presents the results from a series of experiments that examined diffusive O2 supply to submerged organs and the resulting O2 dynamics in completely and partially submerged M. brownii (Hook. f.) (syn. Haloragis brownii; Moody & Les 2007) in both a field situation and in laboratory experiments. M. brownii is an Australian wetland endemic, which can grow in moist soil on wetland fringes with its shoot completely emergent; however, it is more commonly found in areas where, for large parts of the year, shoots are partially or completely submerged by water to depths of 1 m (Marchant et al. 1987; Romanowski 1998). When partially or completely submerged, M. brownii has the ability to grow extensive stem-borne aquatic adventitious roots (Rich et al. 2011; Rich, Ludwig & Colmer 2012), and during lengthy submergence, M. brownii will also develop dissected aquatic adapted leaves (Marchant et al. 1987) to enhance underwater photosynthetic rates (Rich et al. 2011). Due to the relatively high porosity of M. brownii stems (Rich et al. 2011), we hypothesized that plants would show significant connectivity between aerial and submerged portions of the stem, resulting in higher internal O2 in partially submerged plants than in fully submerged plants, due to diffusive movement of O2 down the stems of partially submerged plants. We expected this to be particularly evident pre-dawn, when underwater photosynthesis was not an O2 source and dissolved O2 in the water column would be depleted by net system respiration during the night, so that diffusive atmospheric O2 entry might then be the major source to partially submerged plants.
MATERIALS AND METHODS
Study site and plant materials
An ephemeral wetland near Albany, Western Australia (S 35.0779°, E 117.9267°) was chosen as the site for all in situ studies and as a collection point for plants used in laboratory and anatomical studies as it has a large population of M. brownii (Hook. f.), which is inundated from July to November every year. M. brownii is a wetland species tolerant of several months of inundation and grows an extensive stem-borne aquatic adventitious root system when completely or partially submerged (Rich et al. 2011, 2012). Plants for laboratory work were collected as cuttings, 3–4 h after sunrise on the day they were to be used and kept in aerated water from the wetland. Cuttings to be used in laboratory O2 dynamics were wrapped individually in paper towel dampened with wetland water, placed in plastic bags within an insulated cool-box and transported the same day to Perth, Western Australia. These were then maintained in an aquarium containing aerated water from the wetland, in a 15/20 °C night/day phytotron. Plants for biomass measurements were collected intact and separated into constituent tissue types; tissues were oven dried for 72 h at 60 °C in Perth.
Plant O2 dynamics –in situ field studies
Intra-plant O2 dynamics in aquatic adventitious roots and stems of M. brownii were followed in situ for plants growing in the wetland near Albany. Two, three-channel underwater picoammeters with built-in data loggers (PA3000UP-OP, Unisense, Aarhus, Denmark) and 50 µm tip diameter O2 microelectrodes (OX50-UW, Unisense) was used. Micromanipulators with microelectrodes were mounted on aluminium stands fixed in the peat bottom, and the microelectrodes were positioned using changes in signal to detect the surface of roots or stems following the procedure of Borum et al. (2005) and Pedersen et al. (2006). Oxygen microelectrodes were inserted approximately 150 µm into the aquatic adventitious roots and 250 µm into the stem from which the roots originated. Water depth was ∼46 cm, and the submerged stem and aquatic roots measured were at the mid-water column, and so were at least 23 cm under water, but total length under water would have exceeded 23 cm as the stems were certainly not vertical (regrettably was not measured) and the path length for O2 to move along roots was in addition to the distance along the stem, as aquatic roots were measured ∼10 cm from the root/shoot junction. Two replicate measurements were taken in plants that were either completely submerged or in plants with part of the shoot in air.
Key environmental parameters were measured at the wetland during the period of in situ O2 measurements. Photosynthetically active radiation (PAR) and temperature were recorded using pendant loggers (Hobo Pendant Temp/Light Data Logger UA-001–08, Onset Computer Corporation, Pocasset, MA, USA). Diel fluctuations in pH were logged (LI-1400 Data Logger; Li-Cor Biosciences, Lincoln, NE, USA), and available CO2 was calculated using the temperature and pH data and the formulae of Mackereth, Heron & Tailing (1978). Data were recorded from midday throughout the dark period until the following midday.
Plant O2 dynamics – laboratory
Changes in O2 concentrations were measured in partially submerged stems of M. brownii when under controlled conditions. Partially submerged shoots (∼0.4 m in length) were collected in the field and the submergence level noted, so these shoots could be again submerged to the same level in the laboratory. These shoots then had the O2 concentration around the aerial portion manipulated while internal pO2 was monitored in both submerged and aerial portions of stem, and in aquatic roots.
The shoots to be measured were transferred to a shallow tank (length 0.8 m) made from 150 mm diameter PVC pipe sliced lengthways in half, with caps at each end. The shoot was partially submerged in deoxygenated 0.1% (w/v) agar solution containing in mol m−3: Ca2+ 0.50; Mg2+ 0.25; Cl- 1.00; SO42− 0.25; K+ 1.00, therefore, any O2 measured within the submerged stem or aquatic roots was derived from the shoot, via movement through the aerenchyma. Stem and aquatic root tissues were held in place (for electrode insertion) on Perspex stands using putty (BlueTac, Bostick, Wauwatosa, WI, USA). The aerial portion of the stem was enclosed in a Perspex cylinder, which contained gas entry and exit tubes so the atmosphere within could be easily adjusted. Using a micromanipulator (MM5, Märzhäuser, Wetzlar, Germany), Clark-type O2 microelectrodes with tip diameter of 25 µm (OX25, Unisense) were inserted into an aquatic root and into aerial and submerged regions of the same stem. The distance between the two stem electrodes varied between 75 and 110 mm. A fourth microelectrode was used to monitor O2 concentrations within the aerial cylinder.
After the initial set up, the shoot was left to stabilize. Once quasi-steady state readings were achieved, the atmosphere around the aerial portion of the shoot was flushed with N2 to remove all O2, and left until microelectrode readings stabilized (approximately 3 h), then N2 was replaced with an air/O2 mixture to provide O2 at approximately twice the ambient level (35.7 ± 0.2 kPa O2). During all gas manipulations, the signal of electrodes was logged every 10 s on a computer via a picoammeter (PA2000, Unisense A/S) and an analogue to digital converter (ADC-20, Pico Technology, St Neots, UK). Four replicate plants were measured and experiments were conducted at 18 °C in the dark to prevent any endogenous O2 production.
Rates of O2 diffusion through stems of varying porosity were ascertained to test the relationship between porosity and diffusion. Stem segments of 15 mm containing one or no nodes were partially inserted into a gas-tight 67 mL cylindrical, transparent Perspex cuvette, and held in place using putty (BlueTac, Bostick). The lid of the cuvette was fitted with a syringe port for gas inlet and outlet needles and a mount for a Clark-type O2 minielectrode (OX500, Unisense). The cuvette was flushed with N2 until microelectrode readings indicated that all O2 had been removed. To prevent transfer of gas across the cuticle and to help prevent the cut end of the stem drying out, the cuvette was submerged so only 3 mm of stem segment protruded above the water surface. Diffusion of O2 through the stem segment into the cuvette was logged using the same O2 measurement system as outlined previously. The porosity of internodal and nodal regions of the stem segments was determined as outlined next.
Underwater net photosynthesis
Underwater net photosynthesis of plant tissues was measured as net O2 production in sealed glass bottles mounted on a rotating wheel incubator (Colmer & Pedersen 2008). The glass bottles (50 mL) contained submergence solution (see above). Measurements were started at approximately 50% of air equilibrium for O2 (solution pre-bubbled with N2 and air in 1:1 volumes), with KHCO3 injected into the mixed solution just prior to filling the bottles to achieve 500 mmol m−3 of available CO2 at pH 6 (Stumm & Morgan 1996). This CO2 concentration was chosen as it is close to the level needed for CO2-saturated underwater net photosynthesis (Rich et al. 2011), and corresponded to the highest concentrations measured during the morning at the Albany collection site (Fig. 1). Tissue (two–three leaves or eight 50 mm root segments or three 30 mm stem segments) was placed into bottles with two glass beads (2 mm diameter) to facilitate mixing. The bottles were immediately mounted in the incubator, with bottles without tissues serving as blanks. Following incubations of approximately 1.5 h, dissolved O2 concentrations in the solutions were measured using a minielectrode with protection cap (OX-500, Unisense A/S) connected to a picoammeter (PA2000, Unisense A/S). Measurements were conducted at 17 °C with PAR of 430 ± 7 µmol m−2 s−1. The projected area of leaves was measured using a leaf-area meter (Li-Cor LI-3000). Stem and aquatic root diameters were measured using digital callipers and their surface areas were calculated using the formula for a cylinder.
Tissue characterization – anatomical, histochemical, chlorophyll and porosity analyses
Stems were hand sectioned using a razor blade and stained with Toluidine Blue (0.1% w/v Toluidine Blue in water) to augment visualization. Other sections were stained with phloroglucinol-HCl to visualize lignified cell walls (sections mounted into a filtered, saturated solution of phloroglucinol in 20% v/v HCl), Sudan red 7B to visualize suberins (sections stained for 300 s in a filtered, saturated solution of Sudan red 7B in 70% v/v ethanol, rinsed in 50% ethanol and mounted in 50% v/v glycerol) or aniline blue which reveals callose under ultraviolet (UV) excitation (sections mounted into 5% w/v aniline blue in 0.067 mol m−3 K2HPO4 at pH 8.2). Sections were viewed with a light microscope equipped with a UV filter set (Zeiss Axioskop2, Carl Zeiss Pty. Ltd. Hamburg, Germany). Images were captured with a digital camera (Zeiss AxioCam MRc Rev.3. Carl Zeiss Pty. Ltd.) and manipulated for light levels, contrast and brightness using the Zeiss software package AxioVision Rel. 4.6 (Carl Zeiss Pty. Ltd.). This software package was also used to measure size of various cells (longest measurement across a cell).
Gas spaces (aerenchyma and other intercellular spaces) as a percent of total sectioned area in micrographs of transverse sections of roots or stems were calculated. Sections were photographed on a stereoscope (Zeiss Stemi 2000-C Carl Zeiss Pty. Ltd.) and the public domain software ‘ImageJ 1.32j’ (NIH 2005) was used to determine the percentage of the total area of each section occupied by gas-filled spaces (i.e. providing an estimate of total porosity).
Total chlorophyll was extracted from freeze-dried and ground tissues (10–20 mg dry mass) in cold, 100% v/v methanol (1.25 mL) for 30 min, in darkness (Wellburn 1994). After centrifugation at 9300 g for 10 min at 4 °C, supernatants were collected for analysis. Concentrations of chlorophylls a (Chl a) and b (Chl b) were determined by measuring absorbance of the samples at 665.2 and 652.4 nm, using a quartz cuvette in a UV-visible spectrophotometer (model 1601, Shimadzu, Tokyo, Japan), and the equations from Wellburn (1994).
The theoretical expected flux (J) of O2 through 15 mm long straight stem segments of uniform porosity was calculated as:
where Co is the O2 concentration at the source (0.276 g mm−3 at 18 °C), Do is the diffusion coefficient of O2 in air (at 18 °C, 20.6 mm2 s−1), ε is the fractional porosity (a value between 0 and 1) and L is the length (mm) of the stem segment (note: this equation does not include any O2 consumption by the cells along the diffusion path). Measured fluxes were plotted and a correlation analysis to the theoretical fluxes was made in GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA).
Statistical analyses were undertaken using GenStat 10.0 (VSN International, Hemel Hempstead, UK).
Completely and partially submerged stems show similar diurnal patterns of internal O2 concentration (at least when measured well below the water surface)
There was large diurnal fluctuation in pO2 within the submerged organs of M. brownii, a pattern mirrored by the pO2 of the water column (Fig. 1a,b). In partially submerged plants, pO2 during late afternoon was higher in the submerged portion of the stem and in aquatic roots than in the stem portion in air (Fig. 1b). The highest concentrations of dissolved O2 were also recorded for the water column in the late afternoon (Fig. 1a,b). At night, when all O2 was sourced exogenously, pO2 decreased in all submerged tissues and the water column, reaching almost anoxic levels just prior to dawn (Fig. 1a,b). This pattern was reversed for dissolved CO2 levels, which peaked at dawn (Fig. 1d). Conversely, aerial stem portions of partially submerged plants did not show these large fluctuations due to venting of O2 into the atmosphere during the day and diffusive uptake of O2 during the night (Fig. 1b). The hypothesis that submerged organs of the partially submerged plants would maintain a higher pO2 at night than those of the completely submerged plants, owing to diffusive transfer of atmospheric O2 from the aerial portions of the plant via the aerenchyma, was not evident; pO2 of submerged stems and aquatic roots (at least at the position measured in the mid-water column) was very similar in both completely and partially submerged plants (Fig. 1a,b).
Stems have high internodal porosity but nodes have lower porosity and therefore more resistance to O2 diffusion
The biomass allocation of partially and completely submerged plants differed from that of completely emergent plants (Table 1). Sediment roots are only a small part of the biomass in flooded plants, appearing to be replaced by aquatic root biomass, which were most prolific in the partially submerged plants. Stems are the major constituent of the biomass in this species, regardless of growth condition (Table 1). In the partially submerged plants, stems reached lengths of over 1 m with many nodes. Leaves develop at the nodes in an opposite or alternate configuration, with aquatic roots tending to emerge near to the petiole bases. Internodal lengths varied from 6 to 53 mm, with longer internodal distances occurring in the more deeply submerged stem sections. Both aerial and aquatic adapted leaves had higher concentrations of Chl a than stems and aquatic roots (Table 1), and this was reflected in the significantly higher rates of underwater net photosynthesis in aquatic adapted leaves (Table 1). Both stems and aquatic roots of M. brownii contained large amounts of gas space; in partially submerged plants, aerial stems had 6.3 ± 0.6% gas space per cross-sectional area, while submerged stems in both completely and partially submerged plants had more gas space (17.9 ± 0.6%). Aquatic roots also had high porosity, which did not differ significantly between partially and fully submerged plants (24.3 ± 1.1% gas space per cross-sectional area).
Table 1. Tissue partitioning (% total dry mass, DM), chlorophyll a concentration (Chl a) and underwater net photosynthetic rate of organs from field-collected Meionectes brownii
Chl a (mg g−1 DM)
Underwater net photosynthesis (µmol O2 m−2 s−1)
Completely emergent (% total DM)
Partially submerged (% total DM)
Completely submerged (% total DM)
Plants growing under different conditions varied in mean total DM: completely emergent (1.2 ± 0.27 g), partially submerged (1.9 ± 0.58 g) and completely submerged (1.3 ± 0.12 g). Underwater net photosynthetic rates were measured as O2 production in closed vials at 17 °C with 500 mmol m−3 dissolved CO2 and PAR of 430 ± 7 µmol m−2 s−1. Mean values are shown (±SE) for percentage of total DM of organs from completely emergent, partially or completely submerged plants. n = 10 for DM and n = 4 for Chl a and underwater net photosynthesis. Superscripts indicate significant differences at P < 0.05 (Fisher's LSD; comparisons across each organ type for DM and down columns between all organs for Chl a and underwater photosynthesis).
b.d., below detection, PAR, photosynthetically active radiation.
22.2 ± 1.6a
10.5 ± 1.3b
4.5 ± 0.81a
53.1 ± 3.8a
5.7 ± 0.8b
0.77 ± 0.22b
23.9 ± 3.4a
5.9 ± 1.2b
5.9 ± 1.3b
25.5 ± 4.1a
15.9 ± 7.1a
0.87 ± 0.24b
0.17 ± 0.00a
43.5 ± 6.4a
60.3 ± 8.3b
1.3 ± 0.22b
0.20 ± 0.05a
9 ± 1.4a
17.9 ± 4.4a
4.0 ± 0.30a
3.42 ± 0.80b
To examine the apparent resistance to O2 movement through the stems of partially submerged plants, cuttings from the field were used in laboratory experiments in which the O2 concentration around aerial portion was manipulated and changes in pO2 in aerial and submerged tissues were recorded (Fig. 2; Table 2). O2 movement from the aerial portion to the submerged portion of stems of cuttings in controlled conditions occurred rather slowly (aerial stem and submerged stem measurement positions were separated by 75–110 mm). When the atmosphere around the shoot portion in air was flushed with N2, the pO2 in the aerial tissue responded almost instantly, dropping to near 0 kPa, clearly illustrating that even in darkness there is extensive gas exchange between the atmosphere and the shoot. In contrast, the submerged part of the stem and aquatic root that was in stagnant de-oxygenated agar responded far slower, not reaching 0 kPa even after several hours (Fig. 2; Table 2). When a high O2/air mixture was flushed around the aerial portion of the shoot, again the aerial tissue responded almost instantly with increased pO2; whereas the lag time was substantial for the submerged portion of stem and for the aquatic root, in which pO2 increased and reached a quasi-steady state after 6–8 h. The submerged stems and aquatic roots in deoxygenated agar had 52 and 29% of the pO2 values of the aerial stems, respectively (Table 2).
Table 2. Quasi-steady-state internal O2 partial pressures (pO2) in organs of partially submerged Meionectes brownii plants
Gas provided to aerial portion of shoot
Field-collected M. brownii shoots with attached aquatic roots were partially submerged in a deoxygenated 0.1% (w/v) agar solution. O2 microelectrodes were inserted into both submerged and aerial portions of stems and into aquatic roots (distance between the two stem electrodes was 75 to 110 mm and contained several nodes). The O2 concentration around the aerial portion of the shoots was manipulated while internal pO2 was monitored. Values are means ± SE (n = 3) when pO2 was at a quasi-steady state, over the last 30 min prior to the gas treatment being changed, or from 3 h after the last treatment. Measurements were conducted at ca. 18 °C and microelectrode drift due to temperature changes (of at most 3 °C) were corrected. Measurements were conducted in darkness to prevent endogenous O2 production. Superscripts indicate significant differences at P < 0.05 (Fisher's LSD; comparisons down each column, i.e. each gas mixture).
21.3 ± 0.54a
0.0 ± 0.0a
35.7 ± 0.26a
20.6 ± 0.92a
0.0 ± 0.0a
35.7 ± 0.23a
10.7 ± 1.6b
1.2 ± 0.53b
18.6 ± 1.12b
6.9 ± 1.08c
0.8 ± 0.13b
10.5 ± 1.75c
Stem aerenchyma is interrupted at nodes
Internodal sections of stems from partially submerged M. brownii had high porosity; however, at the nodes, the porosity was usually lower, with around a quarter of observed nodes having less than 5% gas space per cross-sectional area (Fig. 3). The lower porosity was due to the aerenchyma being interrupted near the nodes (Fig 4a,b). Lower porosity was related to the emergence of organs such as leaves, aquatic roots and new stems from the nodes, with aerenchyma interruption being more severe for nodes from which more organs had emerged (Fig. 5). This association did not hold where a new stem emerged, which was at least 80% as thick as the main stem, as in such cases, the stem aerenchyma was inevitably completely interrupted (data not shown; n = 12). While some nodes had only a single leaf emerging, many nodes supported the emergence of a lateral stem and several aquatic roots, as well as a leaf (Fig. 4c). As leaves grow in an alternate or opposite configuration, the aerenchyma is interrupted at least every few nodes. Nodal interruptions occurred in both aerial and submerged stems.
Anatomical and histochemical observations showed no evidence of diaphragms at the stem nodes, and that in addition to the emerging organs themselves, the material interrupting the aerenchyma near the nodes was not callose, but rather consisted of cells that were not lignified or suberized (data not shown). These cells were smaller (45.7 ± 1.7 µm diameter, n = 50 from 10 sections) and more loosely packed than surrounding cortical cells (95.6 ± 3.7 µm diameter), and like those surrounding cells, contained plastids (Fig. 4d,e). Although the cells that interrupt the aerenchyma were loosely packed, the interruptions could be a couple of mm thick (2450.9 ± 445.8 µm, n = 5; Fig. 4f), and therefore create an impediment to O2 diffusion.
Nodal porosity correlates with O2 diffusion rate through stems
The interruptions to aerenchyma at the nodes of the submerged stems from partially submerged M. brownii influenced the capacity for gas diffusion along these stems. A strong relationship (r2 = 0.76, P = 0.0001) between theoretical O2 flux and nodal porosity was found when diffusion rates were measured through 15 mm stem segments containing no more than one node (Fig. 6b). The relationship of internodal porosities (taken 5 mm above the node) to the theoretical O2 flux was far weaker (r2 = 0.27, P = 0.06; Fig. 6a).
The emergence of M. brownii shoots above water offers only modest advantage to the O2 status of deeply submerged portions of these plants as resistance to O2 movement along the stem is relatively high, because of interruptions of the aerenchyma near each node (Fig. 4). Little information exists regarding interruptions to lacunae, with the exception of nodal diaphragms, which offer no significant impediment to diffusive gas movement (Sorrell & Dromgoole 1987; Armstrong & Armstrong 1988; Schuette, Klug & Klomparens 1994; Sorrell, Brix & Orr 1997). Occlusion of petiole aerenchyma through the growth of cells in the petiole wall forming a ‘spongy mass’ has been recorded in the floating leaf genera Nuphar and Brasenia (Sculthorpe 1967). Rhizome aerenchyma blockages have been studied in Phragmites australis, with these being attributed to either callus growth in response to pathogen attack (Armstrong et al. 1996b), or formation of tylosoid-like cells at rhizome nodes in response to internodal breakdown (Armstrong & Armstrong 1988). Unlike the above cases, in M. brownii stems, the aerenchyma interruptions are only in nodal regions, occurring in both aerial and submerged portions of stems. The extent of nodal interruption appears to correlate with the number of organs (leaves, aquatic roots or stems) emerging at any particular node (Fig. 5), and the opposite or alternate arrangement of leaves along the stem results in aerenchyma becoming interrupted every few nodes. Although no other record of stem aerenchyma being interrupted due to organ emergence could be found in the literature, this phenomenon has been described in aerenchymatous roots. In maize (Zea mays), emerging lateral roots are often surrounded by layers of cortical cells, reducing porosity of the primary root (Bouranis et al. 2006), and in Typha glauca, cortical cells were continuous surrounding emerging lateral roots, although the root cortex was highly aerenchymatous in other areas (Seago & Leland 1989). It seems likely that the interruptions to M. brownii stem aerenchyma are a combination of the emerging organ itself and the presence of numerous small, loosely packed, cortical cells in the areas surrounding the new organ (Fig. 4).
The apparent high resistance to diffusion down the stems of partially submerged M. brownii raises the question of the O2 source(s) to the submerged tissues/organs. Tissues near the water surface would benefit from the aerial connection, although nodes appear to significantly reduce O2 movement even over short stem lengths (cf. Fig. 2, with six nodes over 80 mm). If potential aerated path lengths are calculated using the equation ΔCo = ML2/2De[see Armstrong 1979; where L is length, ΔCo is O2 concentration at the source, M is respiratory demand (calculated from respiration measurements taken alongside photosynthesis measurements) and De is effective diffusion coefficient, i.e. diffusion coefficient of O2 × fractional porosity], then a stem with porosity of 17.7% (mean of the internodes) would have a potential aerated length of 0.74 m, and a stem with the mean nodal porosity (8.2%) would have a path length of 0.50 m. These estimates are indicative only as they do not account for the variation in internode and nodal porosity down an M. brownii stem, nor tortuosity, nor the O2 demand of the roots. In the field, however, stem tissues at ∼23 cm below the surface (with a diffusion path length likely longer than depth alone as stems were not vertical) did not receive additional O2 when the shoot tip was emergent, so the above calculation highlights that presence of occluded nodes (see Results) must have substantial negative effects on O2 movement down the stem. Our laboratory measurements of O2 movement (Fig. 2; Table 2) demonstrated that such nodes of low porosity provided a significant impediment to O2 movement. These laboratory results, in combination with our observations that ca. 23% of the nodes we examined (n = 52) were completely occluded and as stems possess 30–50 nodes per m, then stems would likely contain several very low porosity nodes in series (e.g. 10–15 such nodes per m) and the cumulative resistance of these likely restricted internal aeration along stems.
If the aerial connection is not an effective source of O2 to these deeper submerged organs, then, as in completely submerged plants (Sand-Jensen et al. 2005), O2 must be sourced through a combination of underwater photosynthesis and diffusion from the water column (Fig. 1). Rates of underwater net photosynthesis in M. brownii aquatic adapted leaves are >15-fold higher than those of submerged stems and aquatic roots (Table 1). However, given the time needed for O2 movement down the stems (Fig. 2) and the growth of aquatic leaves predominantly near the water surface, O2 produced via photosynthesis in these more apical aquatic leaves also would probably not move readily into deeper submerged stem sections, or into the sediment roots which could be up to 1 m away (the apparent depth limit observed for for M. brownii in field surveys) (Marchant et al. 1987).
In the present field study, water column O2 was never higher than stem or aquatic root pO2 during the day, suggesting that underwater shoot (aquatic leaves) and aquatic root photosynthesis was the primary daytime source of O2 to these organs. During the dark period, tissue pO2 echoed that of the water column, suggesting that the water column acts as a diffusive O2 source once underwater photosynthesis ceases; the steady decline in water column O2 through the night would be due to O2 consumption from net system respiration, which in the wetland used in our study was probably driven by the dense stands of M. brownii. By dawn, both water column and plant O2 levels were low (>3 kPa); however, as soon as the sun rose, plant pO2 increased, with the water column O2 slowly emulating this rise, presumably as O2 diffused out of plant tissues (or was produced by cyanobacteria and algae). Night-time respiration also resulted in dissolved CO2 concentrations being highest at dawn (Fig. 1d), presumably allowing shoots and aquatic roots to achieve relatively high rates of underwater net photosynthesis during the morning. In the late afternoon, diffusive entry of O2 from the water column may again become important to these organs in addition to any endogenous production, particularly when low CO2 availability reduces underwater photosynthetic rates, and during the night inwards diffusion from the water column would supply O2, albeit at a declining rate as the water becomes depleted in O2. In short, partially submerged M. brownii plants act similarly to completely submerged plants, sourcing most of their O2 via inwards diffusion from the water column and endogenous production from underwater photosynthesis.
Roots in anoxic sediment acquire O2 via diffusion from the shoot base through aerenchyma and intercellular gas-filled spaces (Beckett et al. 1988; Armstrong & Drew 2002). If within the stem the resistance to diffusion is high or if the lacunal pathway is long, then O2 at the stem base would be low, so that adequate aeration of the roots would be compromised and, therefore, the size of the root system reduced (Armstrong 1979; Schuette et al. 1994). In M. brownii, the size of the sediment root system is significantly reduced from 24% of the total biomass in completely emergent plants to only 6% in submerged plants (Table 1), although total root biomass actually increases in submerged plants through the extensive growth of aquatic roots. Biomass allocation to both the stems (ca. 50%) and the leaves (ca. 20%) remains the same regardless of submergence level. The diffusion limitation found in the stems of partially submerged M. brownii would likely restrict the O2 supply of the sediment roots, which in turn provides a possible explanation for the sediment root dieback observed in this species during flooding. By contrast with the sediment roots, new aquatic roots can source O2 both endogenously via root photosynthesis (Rich et al. 2011) and exogenously through diffusion from the water column.
In previous studies of in situ O2 dynamics in submerged plants (Greve et al. 2003; Sand-Jensen et al. 2005; Borum et al. 2006; Pedersen et al. 2006; Holmer et al. 2009), and in the current study M. brownii, there was a strong decline in internal pO2 as light levels decrease towards evening and a slow decline during the dark period, with several species, including M. brownii, having very low internal O2 concentrations or even becoming anoxic by dawn (cf. Thalassia testudinum, Holmer et al. 2009). In contrast to these previous studies, which largely examined marine systems and showed declines in water column O2 to no less than 75% air equilibrium, we recorded strong declines in water column O2 levels overnight, with dawn measurements being ca. 15% of air equilibrium and not increasing to above shoot pO2 until late afternoon. Therefore, while diffusion from the water column is likely of importance at night, higher internal plant pO2 in the early mornings and the day results in diffusion from the water column not being possible for M. brownii, indicating a reliance on underwater photosynthesis during the early morning, but it is therefore intriguing that the internal O2 transport via the stem aerenchyma in this species is apparently relatively poor (e.g. compared with rice, see Introduction).
In conclusion, a shoot aerial connection in partially submerged M. brownii was of surprisingly little benefit to the O2 status of submerged tissues beyond the first several stem nodes, although this connection presumably enhances the carbon balance of the entire plant through aerial photosynthesis, and emergence would also enable reproduction. Deeper organs/tissues of partially submerged M. brownii, like fully submerged plants, relied on endogenously produced O2 and on entry of exogenous O2 from the water column. An aerial connection is doubtless of high importance to the O2 status in many wetland species which undergo short-term shallow submergence or respond to submergence with an elongation response (i.e. rice, Rumex palustris; Bailey-Serres & Voesenek 2008; Colmer & Voesenek 2009); however, we hypothesize that for some species, which are tolerant of extended periods of partial submergence, possibly with periodic complete submergence, the aerial connection may be one of a suite of mechanisms for sourcing O2. The nodal anatomy of most plants is not well documented, or is highly varied between cultivars (i.e. Oryza sativa: Higaskmura 1969; Catling 1992; Steffens, Geske & Sauter 2011), and consequently the diffusion potential through nodes of most species is unknown. It is likely that some other wetland species also may have interruptions to stem aerenchyma. The present study on M. brownii not only cautions against generalized assumptions regarding plant aeration, especially across species from differing ecological niches, but also highlights the need for more studies of partially submerged plants across a broader range of species to gain a greater understanding of internal aeration during flooding.
We thank the Faculty of Natural and Agricultural Sciences at UWA for supporting O.P. under the Distinguished Visitors Program; Bill Armstrong for assistance with the calculation of theoretical diffusion rates and very helpful comments on an earlier version of this manuscript; Kevin Hopkinson from the Department of Water (South Coast Region) for introducing us to the wetland near Albany; Kathryn O'Brien and John Rich for allowing their home to be converted into a field laboratory and accommodation for one week. S.R. acknowledges scholarship and operating support from the Land and Water Resources Research and Development Corporation and the Future Farm Industries CRC.