Flash floods can submerge paddy field rice (Oryza sativa), with adverse effects on internal aeration, sugar status and survival. Here, we investigated the in situ aeration of roots of rice during complete submergence, and elucidated how underwater photosynthesis and floodwater pO2 influence root aeration in anoxic soil.
In the field, root pO2 was measured using microelectrodes during 2 d of complete submergence. Leaf gas films that formed on the superhydrophobic leaves were left intact, or experimentally removed, to elucidate their effect on internal aeration.
In darkness, root pO2 declined to very low concentrations (0.24 kPa) and was strongly correlated with floodwater pO2. In light, root pO2 was high (14 kPa) and primarily a function of the incident light determining the rates of underwater net photosynthesis. Plants with intact leaf gas films maintained higher underwater net photosynthesis relative to plants without gas films when the submerged shoots were in light.
During complete submergence, internal aeration of rice in the field relies on underwater photosynthesis during the day and entry of O2 from the floodwater during the night. Leaf gas films enhance photosynthesis during submergence leading to improved O2 production and sugar status, and therefore contribute to the submergence tolerance of rice.
Complete submergence presents an array of challenges to terrestrial plants, among which internal aeration is paramount (Armstrong, 1979). Paddy field rice is generally tolerant to waterlogging and partial submergence (Colmer, 2003), but lowland rice can also experience flash floods that completely inundate the plants for 10–15 d (Zeigler & Puckridge, 1995). In the rainfed lowlands of India, submergence is considered as the third most important limitation to rice production, after drought at anthesis and weeds (Widawsky & O'Toole, 1990). Internal aeration of rice during submergence has been studied in laboratory experiments (Waters et al., 1989; Colmer & Pedersen, 2008a), and the findings are considered below, whereas in situ (i.e. field) data only exist for deepwater rice (Setter et al., 1987), which elongates substantially to remain in contact with the atmosphere during rising floodwaters, a markedly different situation and response to that of completely submerged lowland rice (Bailey-Serres & Voesenek, 2008; Bailey-Serres et al., 2010).
The flooding environment experienced by terrestrial plants invokes constraints of greatly restricted gas exchange and low light in comparison to when shoots are in air (Mommer & Visser, 2005; Colmer et al., 2011). Diffusion in water is 104-fold slower than in air, which severely impedes O2 and CO2 exchange between the plant and the environment (Armstrong, 1979). Moreover, O2 solubility in water is low and natural diurnal fluctuations of O2 in the floodwater can result in hypoxic conditions at dawn following a night-time period in which net O2 consumption has occurred as a result of system respiration (Setter et al., 1988). During the day, O2 produced by underwater photosynthesis by plants and microalgae can again increase the O2 concentration that peaks in the late afternoon (Ram et al., 1999). As a consequence of the tight coupling between respiration and underwater photosynthesis, CO2 in the floodwater follows the opposite pattern of O2, with the highest concentrations at dawn and the lowest concentrations during the late afternoon (Sand-Jensen & Frost-Christensen, 1998). In addition, floodwaters are also often turbid because of suspended inorganic particles and microalgae (Setter et al., 1987), and this will further decrease the potential for underwater photosynthesis of rice (and other plants) when completely submerged (Das et al., 2009).
Plants can photosynthesize when under water provided that both the light and CO2 levels are sufficient. In air, CO2 enters the tissue via open stomata, whereas, under water, the stomata are hypothesized to close (Mommer & Visser, 2005), or, at least, when surrounded by water, the high boundary layer resistance greatly slows down gas exchange. Therefore, CO2 first has to overcome the resistance caused by the aqueous diffusive boundary layer via slow molecular diffusion and then subsequently cross the cuticle which also adds significantly to the total resistance to CO2 uptake (Mommer & Visser, 2005). Although most floodwaters contain CO2 concentrations above air equilibrium levels, underwater photosynthesis is still restricted by low CO2 supply caused by the much higher resistance for leaf uptake when under water (Colmer et al., 2011). When submerged, some terrestrial wetland plants acclimate to facilitate gas exchange with the water by the production of new ‘semi-aquatic’ leaves which are thin, as well as having reduced cuticles and rearrangement of chloroplasts closer to the epidermis, all resulting in lower resistance to CO2 diffusion to chloroplasts (Mommer & Visser, 2005; Mommer et al., 2005). This strategy requires investment in new acclimated leaves. Some terrestrial wetland plants, including rice, however, can photosynthesize under water with their pre-existing aerial-type leaves, as these retain a thin gas film when submerged (Raskin & Kende, 1983; Colmer & Pedersen, 2008b). Leaf gas films greatly facilitate gas exchange with the floodwater (Colmer & Pedersen, 2008b; Pedersen et al., 2009; Pedersen & Colmer, 2012) and, as no period of acclimation is needed, this feature functions well during recurring floods, such as in the tidal zone (Winkel et al., 2011). The increased gas exchange with the floodwater caused by the presence of leaf gas films results in improved CO2 uptake and underwater net photosynthesis during the day and improved internal aeration during the night, for submerged plants (Colmer & Pedersen, 2008b; Pedersen et al., 2009; Colmer et al., 2011; Winkel et al., 2011; Pedersen & Colmer, 2012).
Underwater net photosynthesis and internal aeration of paddy rice have been studied under laboratory conditions. Waters et al. (1989) described the diurnal fluctuations of radial O2 loss (ROL) from roots of completely submerged rice, with higher ROL during light periods than when shoots were in darkness. They also showed that root tip O2 declined to very low levels during dark periods, so that root growth ceased. When light was again available, root growth commenced as photosynthetic O2 moved from the shoots via the aerenchyma to the root tips. More recently, the crucial role of leaf gas films for internal aeration, underwater net photosynthesis and growth of submerged rice was demonstrated in controlled-environment experiments. Pedersen et al. (2009) showed that the pO2 (oxygen partial pressure, kPa) near the root tip dropped to critically low values (0.1 kPa) in the dark on experimental removal of the leaf gas films from submerged rice, and, in the light, root pO2 also decreased on removal of the gas films as a consequence of lower net photosynthesis under water. Leaf gas films, and the resulting rates of underwater net photosynthesis, enabled completely submerged rice to grow during 7 d of submergence as well as control plants with shoots in air in a controlled-environment experiment (Pedersen et al., 2009).
In the present study, we conducted the first in situ (i.e. field) real-time measurements of pO2 in rice roots during a submergence event, and our measurements spanned > 2 d. We tested the effect of the removal of leaf gas films on internal aeration and tissue sugar status of submerged rice in a field situation. Moreover, we monitored environmental parameters of floodwater pO2, pH, temperature, alkalinity (and thus calculated the dissolved CO2) and light in order to unravel the effect of the complex relationships between underwater photosynthesis and floodwater pO2 on internal aeration of submerged rice. We tested three hypotheses: (1) in darkness, root pO2 declines on submergence because of constraints in O2 uptake by the shoot from the floodwater; (2) during the day, root pO2 increases as underwater net photosynthesis supplies O2, but internal pO2 might fluctuate as light availability is variable across the day; and (3) leaf gas films improve the internal aeration of submerged rice during the day, as enhanced CO2 uptake promotes photosynthesis, supplying endogenously produced O2, and during the night via enhanced O2 uptake from the floodwater.
Materials and Methods
Plant materials and field site
Root pO2 field experiments were conducted in the wet season (October to November) in the submergence field (bunded field) facilities at the International Rice Research Institute at Los Baños, the Philippines, with the field and soil type described previously (Singh et al., 2009). Rice (Oryza sativa L., cv Swarna-Sub1) was sown in a seedbed in September 2011, and 14-d-old seedlings were subsequently transplanted at 20 cm × 20 cm spacing into a waterlogged paddy field surrounded by bunds to enable submergence to be imposed. Swarna-Sub1 is a dwarf rain-fed lowland Indian variety introgressed with the SUB1A quantitative trait locus that confers submergence tolerance through restricted underwater elongation (Xu et al., 2006). Experiments were commenced 14 d after transplanting, so that plants were 4 wk old (mean height, 28.9 ± 1.1 cm; main stem leaf number, 5.7 ± 0.21; number of tillers, 2 ± 0.21; tallest tiller height, 12.3 ± 5.4 cm; values are means ± SE, n =5). Complete submergence was achieved by flooding with reservoir water (4.8 mM alkalinity; electrical conductivity (EC), 650 μS cm−1; pH 7.9–8.2; water temperature, 26.6–31.9°C). Control plants (nonsubmerged plants) were grown adjacent to the floodable field also in a paddy field.
We also conducted a second field experiment with potted plants from the same batch as stated above, except that these were not transplanted into the field, but instead kept in pots and placed in another paddy field and subsequently flooded with reservoir water (water parameters measured each morning at 10:00 h were, on average, 5.3 mM alkalinity, EC = 650 μS cm−1, pH 7.96 and water temperature of c. 29°C). Pots were 15 cm in diameter and 19 cm deep. The soil type was Maahas (described in Tirol-Padre & Ladha, 2004). There were eight pots with four plants in each; two plants in each pot were brushed with 0.01% (v/v) Triton X-100 in ‘artificial floodwater’ (composition in the section ‘Net photosynthesis under water and in air’) to remove the gas films; the remaining two plants served as controls with gas films intact. The pots were submerged and the plants were harvested after 1, 3, 5 and 7 d of complete submergence. The plants were then divided into tissue samples of leaf blades, sheath/stem and roots.
In situ pO2 dynamics in roots
Root pO2 was measured in situ for plants in the submergence field. O2 microelectrodes (OX-25; Unisense A/S, Aarhus, Denmark), with a tip diameter of 25 µm, were used to measure pO2 inside the roots. Two YSI probes (Pro 1020; YSI, Yellow Springs, OH, USA) were used to monitor floodwater pH, temperature and O2. Microelectrodes and YSI probes were calibrated at known temperature in water at air equilibrium (20.6 kPa O2) and in anoxic water (0 kPa O2) containing 0.1 M sodium ascorbate and 0.1 M NaOH, and the microelectrode signal was corrected to ambient temperature using the procedure of Greve et al. (2003).
Small excavations in the soil were carefully made adjacent to individual plants to expose the upper few cm of the adventitious roots (c. 4 cm below the soil surface, the depth of the shoot base). A spray bottle with floodwater was used to rinse mud off the roots. When the excavation was complete, aluminum stands were fixed in the soil adjacent to the plant and mounted with micromanipulators (MM33; Unisense A/S; Fig. 1a). The O2 microelectrode tip was inserted c. 200 μm into the adventitious root and so was in the cortex, c. 10 mm below the root–shoot junction. Positioning of the microelectrode followed the procedure of Borum et al. (2005). The excavation was refilled with wet soil after positioning of the microelectrode was complete. Nine microelectrodes were placed in nine plants, five of which were controls with leaf gas films intact and four were treatments with leaf gas films removed. Of the nine microelectrodes, four produced reliable daytime traces and three night-time traces, whereas the remaining five were broken or misaligned because of the unstable substrate causing movements of the setup as we worked in the paddy field. The experiment was repeated with nine microelectrodes inserted in the leaf sheath bases (5 cm below the soil surface) of plants in a second field. Five produced reliable daytime traces and three night-time traces of submerged plants, whereas three microelectrodes placed in the sheaths of plants for which the shoots remained in air in a third field were broken because of wind gusts causing plants with shoots in air to move (Figs 2, S2, S3). The leaves of selected plants were brushed with a diluted Triton X-100 solution (0.01% v/v in artificial water) and then rinsed with floodwater, c. 3 h before release of the water into the field, to reduce surface hydrophobicity so that gas films did not form on submergence (cf. Winkel et al., 2011).
To submerge the plants, a water inlet from the reservoir was opened and the field was flooded at c. 15 cm h−1 to a depth of c. 50 cm above the soil, so that all plants were completely submerged. The experiment lasted for 89 h, spanning 4 d, with the plants completely submerged for 48 h, followed by a period of de-submergence when the field was drained. The timeline of the complete experiment was as follows: at 13:00 h of day 1, the plants were chosen and those selected for treatment were brushed with 0.01% (v/v) Triton X-100; microelectrodes were inserted into the plants between 14:00 h and 16:00 h of day 1; at c. 16:00 h, water was let into the field and the plants were completely submerged by c. 18:00 h. Forty-eight hours later, at 18:00 h of day 3, water was drained from the bunded field. The experiment was concluded when the electrodes were removed in the afternoon of day 4.
Water samples were taken for alkalinity measurements using a Gran titration (Stumm & Morgan, 1996) each morning during the submergence period, and light attenuation of the floodwater (at the surface and 20 cm below the surface) was also measured each day using a spherical PAR sensor (US-SQS/L; Walz, Effeltrich, Germany).
Net photosynthesis under water and in air
Underwater net photosynthesis was measured on excised leaf segments with varying dissolved CO2 concentrations. Four replicate leaves (the youngest fully expanded from four different plants) were taken from the control field and used for underwater net photosynthesis measurements. Two 20-mm leaf segments (c. 200 mm2) were excised halfway up the blade. One segment was used as control (leaf gas film present) and the other was brushed with 0.01% (v/v) Triton X-100 solution in artificial floodwater for the ‘no leaf gas film’ treatment. Underwater net photosynthesis was measured at 30°C using 25-ml glass vials with two glass beads added to ensure mixing according to the method of Colmer & Pedersen (2008b), with photosynthetically active radiation (PAR) inside the vials of 760 ± 60 μmol m−2 s−1 (mean ± SE, n =10). Vials without leaf segments served as blanks.
The incubation medium was artificial floodwater based on a general purpose culture medium described by Colmer & Pedersen (2008b). Various amounts of KHCO3 were added to the artificial floodwater, and 0.1 M HCl was used to adjust the pH (6.3–8.7), thus converting to free CO2, in order to achieve the desired levels of dissolved CO2 (20–5000 μM), whilst keeping the alkalinity (sum of alkaline ions, mainly ) constant at 5 mM (Mackereth, 1978).
Following incubations of known durations (30–50 min), the dissolved O2 concentration in each vial was measured using an O2 minielectrode (OX-500; Unisense A/S) connected to a multimeter (MicroSensor Multimeter; Unisense A/S). All leaf samples were freeze dried and the dry mass (DM) was recorded. A relationship between DM and area was established for segments from the same types of leaves, so that the projected area of each leaf segment could be calculated. Four replicates were used for each gas film and CO2 level treatment combination.
Net photosynthesis in air was measured on the most recent fully expanded leaf using an infrared gas analyzer (IRGA) (LI-6400; Li-Cor, Lincoln, NE, USA) at PAR of 750 μmol m−2 s−1 and CO2 of 380 μl l−1 at 30°C between 10:00 h and 11:00 h.
Leaf characteristics (area : DM, tissue porosity, gas film thickness)
To establish the area : DM ratio of the lamina, the mid-sections (c. 20 mm) of the lamina of the youngest fully expanded leaves from 10 plants from the control field were scanned (V70; Epson, Nagano, Japan). The leaf segments were then oven dried for 48 h at 60°C to constant mass and weighed. The individual scanned areas were then printed on paper and cut out, and the areas were measured using a leaf area meter (LI-3000; Li-Cor).
External gas film volume and tissue porosity (percentage of gas spaces per unit volume of tissue) were measured for leaves by determining the tissue buoyancy before and after gas film removal, followed by vacuum infiltration of the gas spaces with water, using the method of Raskin (1983) with equations as modified by Thomson et al. (1990). Leaf laminae were cut into 5-cm segments for the measurements; only mid-leaf lamina segments were used. The leaf segment area was calculated from the area : fresh mass (FM) ratio. The gas film thickness was estimated by relating the gas film volume to the total leaf surface area (i.e. both sides), as rice has gas films on both the adaxial and abaxial sides of the leaf (Pedersen et al., 2009).
Tissue sugar concentration and total leaf chlorophyll concentration
Chlorophyll concentration was measured in the leaf segments used for underwater net photosynthesis, as well as on leaf segments taken from other plants in the experimental field. The leaf segments were flash frozen in liquid N2, freeze dried for 48 h and stored in a freezer at −80°C until analysis. Chlorophyll was extracted in 80% acetone at 5°C for 12 h in darkness, and light extinction in extracts was measured at 652 nm on a spectrophotometer (UV-VIS 1800; Shimadzu, Nishinokyo, Kyoto, Japan). Chlorophyll concentrations were calculated from the light absorption using the equations of Mackinney (1941).
Tissue sugar concentrations were measured on leaf blades, sheath/stem and root samples from the second field experiment using potted plants. The tissue samples were flash frozen in liquid N2, freeze dried for 48 h and stored in a freezer at −80°C until analysis. Sugars were extracted from tissue samples boiled twice in 80% ethanol with reflux for 20 min. Total sugar levels in the extracts were measured using anthrone (Fales, 1951) and a spectrophotometer (UV-VIS 1800; Shimadzu).
GraphPad Prism 5 (GraphPad Software Inc., http://www.graphpad.com) was used to fit models and for data analysis, and two-way ANOVA with Bonferroni post hoc test was used to compare the means of the differences in sugar and chlorophyll concentrations for plants with and without leaf gas films. A modified nonlinear Jassby & Platt (1976) equation was used to model data for the CO2 response curve (Fig. 3) and for root pO2 as a function of light (Figs 5, S3). A linear least sum of squares was used to fit the data on root pO2 as a function of floodwater pO2 (Fig. 4). Student's t-test was used to test for differences in treatments in the CO2 response curve (Fig. 3) at low nonsaturating CO2 concentrations.
Environmental parameters of the floodwater
Floodwater pO2 followed a diurnal pattern, with the lowest values in the early morning just before sunrise, being well below air equilibrium (5.2 kPa or 62 μmol O2 l−1). During the light period, floodwater pO2 increased throughout the day and peaked just before sunset, reaching 19 kPa or 214 μmol O2 l−1 (just below air equilibrium of 20.6 kPa or c. 236 μmol O2 l−1 at 30°C), and then declined again during darkness. Using the alkalinity (4.83 mM), pH and EC (650 μS cm−1) data for the floodwater in the field, the CO2 concentration in the floodwater was calculated throughout the submergence period. pH was lowest in the early morning (7.9) and highest in the late afternoon (8.22). Thus, CO2 followed the opposite diurnal pattern to that of O2 (low during the afternoon, highest towards the end of the night), but dissolved CO2 was always above the air equilibrium value (at 30°C, c. 11 μM), with values ranging from 54 to 108 μM. Light extinction of the floodwater was −1.75 m−1, resulting in 65% of surface radiation reaching mid-canopy height of the submerged rice plants (depth, 0.25 m).
Leaf porosity and leaf gas film thickness
The porosities of the leaf blades and leaf sheaths were measured, as the amount of gas space within a tissue determines the capacity for the internal diffusion of gases. Porosity for the leaf blades was 9.6 ± 1.2% (n =4) and for the leaf sheaths was 56.1 ± 0.9% (n =4). The leaf gas film thickness was 57.0 ± 5.3 μm (n =4).
Root pO2 dynamics during complete submergence of rice in a field
Root pO2 was measured to determine the in situ O2 status of completely submerged rice in field conditions. Root pO2 was highly variable during the light cycle, with high light intensities corresponding with high values of root pO2 (Fig. 2a). Just before submergence, the root pO2 values of plants with shoots in air and not brushed with 0.01% (v/v) Triton X-100 were 13.1 and 14.4 kPa, and those of plants that had been brushed with diluted Triton X-100 were 9.8 and 11.2 kPa; the brushing and washing resulted in some water covering the sheath–lamina junctions, so that sheath photosynthesis was temporarily reduced (see transient reductions for brushed leaves in Pedersen et al., 2009), which could have impeded O2 entry or lowered internal production. When submerged, during the daylight with periods of high light intensity (noon on the first day of submergence), the root pO2 values of plants with intact gas films were 13.8 and 14 kPa, and, for plants without leaf gas films, the root pO2 values were 10.5 and 11.8 kPa. During the night, root pO2 dropped to very low minimum values of 0.24–0.42 kPa in plants with leaf gas films and below detection (essentially 0.0) for the plant without leaf gas films for which data were available during the night (Fig. 2a). The relationship between internal O2 and light (high internal pO2 during periods with clear skies, lower pO2 in cloudy conditions) and the very low internal pO2 during the night were confirmed in experiments on a second set of plants in a second field with microelectrodes inserted into the leaf sheath bases, 5 cm below the soil surface (Fig. S2).
Light intensities varied as a result of cloud cover with occasional clear skies (Fig. 2b), with a maximum value of 954 W m−2. During the first day of submergence, light intensities varied from 954 to 134 W m−2 in the time span between 08:00 h and 15:00 h, with the minimum value representing < 15% of the maximum light intensity. During this period, root pO2 followed the same pattern, with the highest pO2 values coinciding with the highest light intensities and the lowest light intensities resulting in root pO2 values of < 55% of the maximum. The cloud cover and the resulting intermittent decrease in sunlight caused root pO2 in all the plants to drop by almost 50%.
CO2 response curves of underwater net photosynthesis of rice leaves with or without gas films
Underwater net photosynthesis of rice leaves with or without gas films was measured at a PAR value of 760 μmol photons m−2 s−1 and at CO2 concentrations varying from 20 μM to 5000 μM CO2. The highest CO2 concentration used was not relevant to field conditions, but was included in order to evaluate whether the differences between the two treatments could be removed by the supply of very high CO2, so as to overcome diffusion limitations to CO2 entry. The lower CO2 concentrations used covered the range of those measured in the field floodwater. At 20 μM of free CO2, there was a significant difference between the underwater net photosynthesis by leaf segments with or without gas films: 1.07 ± 0.09 and 0.30 ± 0.06 μmol O2 m−2 s−1, respectively (n =4, one-tailed t-test, P <0.001). At CO2 concentrations similar to those in the field (c. 75 μM CO2), the leaf segments with gas films also showed significantly higher underwater net photosynthesis of 1.53 μmol O2 m−2 s−1, compared with only 0.33 μmol O2 m−2 s−1 for leaf segments without gas films (linear regression of the underwater photosynthesis data between 20 and 250 μM where the data points were close to linear, CO2 saturation not reached). CO2 saturation for leaves with gas films present was reached at c. 1000 μM CO2, and, for leaves without gas films, underwater net photosynthesis was apparently near saturation at the highest CO2 concentration of 5000 μM used (net photosynthesis rates by leaf segments with or without gas films did not differ at this highest CO2 concentration, n =4, one-tailed t-test, P >0.05, Fig. 3).
Taking into account these data on underwater net photosynthesis (Fig. 3), together with the earlier presented data showing that the root pO2 of plants with intact leaf gas films during the first day is higher than that in plants without leaf gas films in the in situ experiment (Fig. 2), it can be suggested that the higher root pO2 is caused by the 4.6-times higher underwater net photosynthesis at ambient CO2 concentrations (c. 75 μM CO2) in the floodwater. Relative to net photosynthesis in air of 33.8 μmol CO2 m−2 s−1, underwater net photosynthesis by leaf segments with CO2 near ambient levels in the field (c. 75 μM free CO2) is 4.5% for leaves with gas films intact and only 1% for those without gas films.
Floodwater pO2 influences root pO2 of completely submerged plants when in darkness
To evaluate the influence of floodwater pO2 on root pO2 status during the night, root pO2 values were plotted against the night-time floodwater pO2 (Fig. 4). There was a positive correlation between root pO2 and floodwater pO2 (Fig. 4, r2 = 0.73).
Light determines root pO2 of completely submerged rice during the daytime
To determine the effect of underwater net photosynthesis and floodwater pO2 on root pO2 status during complete submergence, we correlated the light intensities with root pO2 with a temporal resolution of 15 min throughout the two daylight periods and fitted the data to a Jassby & Platt (1976) model. Because the floodwater pO2 increased throughout the daylight period, we also assessed the possible influence of this parameter by analyzing the residual values from the Jassby & Platt model plotted against the floodwater pO2 values.
There was a positive correlation between root pO2 and light intensity, with varying maximum estimated root pO2 values of plants with and without leaf gas films. Plants with leaf gas films achieved a higher maximum estimated root pO2 value (10.8 kPa, r2 = 0.70, n =2) than that of the plants without gas films (7.0 kPa, r2 = 0.44, n =2) during the daylight periods of the submergence experiment (Fig. 5a). These strong relationships indicate that light is the main determinant of root pO2 in submerged rice during the day, explaining 70% of the variation of plants with gas films and 44% of the variation of plants without gas films.
To further elucidate the role of floodwater pO2 on root pO2 status during daylight, we plotted the root pO2 of plants with gas films from both daylight periods against the light intensities (Fig. 5b). By sorting the data points between morning and afternoon periods, we could visualize the difference in root pO2 as being dependent on light at different floodwater pO2 values, as pO2 in the floodwater increased continually throughout the daylight period, and thus was lower before noon and higher in the afternoon (Fig. 2b). Interestingly, the data points before and after noon were distributed below and above the Jassby & Platt model, respectively, indicating that the model either under- or overestimated the root pO2 values depending on the floodwater pO2. Therefore, we plotted the residual values from Fig. 5(b) against the floodwater pO2 and found a positive linear correlation (Fig. 5d), suggesting that some of the unexplained variation of the Jassby & Platt model could be attributed to the varying floodwater pO2 values. To summarize, 46% (Fig. 5d; linear regression, black line; r2 = 0.46) of the variation in root pO2 of submerged plants with gas films during the daylight, which could not be explained by irradiance, could be attributed to the changes in the surrounding floodwater pO2, meaning that almost one-half of the remaining 30% of the unexplained variation (70% of the variation was attributed to light) could be attributed to floodwater pO2 with a total explained variation of almost 85%. When comparing plants with and without gas films in the first daylight period, it seems that the root pO2 values of plants with gas films are more strongly influenced by floodwater pO2 than are those of the plants without gas films (Fig. 5c; r2 = 0.46 and 0.24, respectively).
Tissue sugars and total chlorophyll concentrations in leaves of rice
Sugar and chlorophyll concentrations during submergence were measured for plants with or without leaf gas films. Total leaf sugar concentration was significantly higher (P <0.001, n =4) in plants with gas films intact for the initial 5 d, but, in both cases, dropped to equally low levels at 7 d (Fig. 6a). Stem/sheath and root soluble sugar concentrations also declined markedly with time of submergence, but, in these tissues, there was no statistically significant difference between plants with or without leaf gas films (Fig. 6b,c). Total leaf chlorophyll concentration declined following submergence with no significant difference between plants with or without leaf gas films (Fig. 6d).
Internal aeration of paddy field rice was studied under field conditions by monitoring pO2 in the adventitious roots of completely submerged plants. In brief, we found that internal aeration during the night relied on a steady O2 flux from the surrounding floodwater into the shoots, and root pO2 declined during the night to hypoxic levels (0–0.42 kPa) at dawn. Internal aeration during the day was controlled mainly by O2 produced in underwater photosynthesis, but also by floodwater pO2 status, resulting in a more complex relationship between root pO2 and the environment. By contrast, our attempt to assess the influence of leaf gas films was less conclusive, with differences in root pO2 between plants with or without gas films only evident during the first day of submergence and, furthermore, was limited by obtaining only two successful replicates for each treatment. In this discussion, we consider the implications of these findings in a general context of the submergence tolerance of terrestrial wetland plants, and compare the response of rice with that of true aquatic plants that have adapted well to the stressors evoked by inundation.
Inundation impedes O2 and CO2 exchange between plant tissues and the environment because of the 104-fold slower diffusion of gases in water compared with that in air (Armstrong, 1979). During the night, submerged plants rely on an inward flux of O2 from the surrounding water to sustain aerobic respiration. Although gas-filled porous tissues hold O2, either from earlier contact with the atmosphere or from endogenously produced O2 from photosynthesis, this is rapidly consumed and/or lost to anoxic soils by ROL from the roots, or to the floodwater if pO2 is below that of the shoot tissues and the plant is photosynthetically inactive. In seagrasses, the O2 reservoir corresponds to only 2–4 min of O2 produced in underwater photosynthesis (Sand-Jensen et al., 2005), and experimental manipulation of the water column has shown that internal pO2 equilibrates with water column pO2 in a matter of 30 min or less (Binzer et al., 2005). After nightfall, tissue respiration and the ongoing O2 loss from the roots result in an O2 gradient from floodwater to shoot, and this gradient drives a continuous flux of O2 from the floodwater and into the shoot and further to the belowground tissues. In the case of rice, there is a linear relationship between root pO2 and floodwater pO2 (73% of the variation in adventitious root pO2 is explained by changes in floodwater pO2; Fig. 4). Extrapolation of the correlation line in Fig. 4 indicates that the adventitious roots might turn anoxic at a floodwater pO2 of 4 kPa. However, the slope of the correlation line in Fig. 4, and thus also the intercept with the y-axis, probably depends on the mixing properties of the floodwater. Work with seagrasses has demonstrated the effect of water column flow velocity on the aeration of belowground tissues in darkness; as the flow velocity increases, the diffusive boundary layers on the leaves decrease, so that the same water column pO2 can sustain a higher rhizome pO2 in agreement with Fick's first law of diffusion (Binzer et al., 2005).
In the present study, the O2 microelectrodes were inserted into the proximal parts of the adventitious roots, c. 1 cm below the root–shoot junction, so that root tips further from the root–shoot junction would have experienced even lower pO2, as some O2 is consumed in tissue respiration and lost via ROL before it reaches the root tips. Consequently, as the proximal parts of the roots approached anoxia towards the end of the night (Fig. 2), the root tips might have turned anoxic for a period of time before sunrise, potentially causing the growth of roots to cease. The cessation of root extension associated with the low apical O2 of submerged rice seedlings during darkness was observed by Waters et al. (1989) in a laboratory experiment. However, this contrasts with laboratory studies on submerged rice showing that root tips can remain well above zero O2 during dark submergence (Colmer & Pedersen, 2008a; Pedersen et al., 2009); however, in these laboratory studies, no strong O2 sink was present surrounding the roots, as compared with the reduced anoxic soil in this field study. In addition, Waters et al. (1989) demonstrated a strong temperature effect; as the temperature increased, the root O2 declined owing to greater consumption in respiration along the diffusion path. In conclusion, pO2 in the roots of submerged rice during darkness is determined by interactive effects of water column O2, tissue respiration (temperature dependent), ROL (from roots as well as buried sheaths), distance from the O2 source and effective resistance to diffusion.
As the sun rises, the internal aeration status of the belowground tissues changes almost instantly and substantially. During the day, light is the main determinant of root pO2 of completely submerged rice, although the floodwater pO2 also influences internal O2 status (Figs 2, 5). In the early morning, root pO2 increased rapidly from hypoxic levels (< 1 kPa just before sunrise) to > 10 kPa in a matter of 2 h (Fig. 2). During that period, floodwater pO2 only increased from 7 to 8 kPa, ruling out a rise in floodwater pO2 as the cause of the observed increase in tissue pO2. Instead, underwater photosynthesis dominates as the source of O2 for root aeration during the day, supported by the relationship between incoming light and root pO2, which resembles an ordinary photosynthesis vs light response curve (Figs 5a,b, S3). Laboratory studies of submerged rice have emphasized the importance of light and the resulting underwater photosynthesis for root aeration, albeit under artificial conditions with roots in deoxygenated agar rather than in soil with a significant O2 demand (Waters et al., 1989; Colmer & Pedersen, 2008a; Pedersen et al., 2009). In situ studies of internodal lacunae of deepwater rice (not completely submerged; Setter et al., 1987), the leaf meristem of the seagrass Zostera marina (Sand-Jensen et al., 2005) and the rhizomes of completely submerged Spartina (Gleason & Zieman, 1981; Winkel et al., 2011) have shown a similarly strong dependence on light for internal aeration. However, in the present study on rice, the variation in incoming light explains 70% of the variation in root pO2, leaving 30% of the variation caused by other factors (plants with gas films, legend of Fig. 5).
Much of the variation in root pO2 not explained by light is caused by variation in floodwater pO2. Early in the morning, when floodwater pO2 is low, a higher proportion of the O2 produced in underwater photosynthesis will diffuse out of the leaves and into the floodwater, which, in turn, will reduce the amount that moves downwards into the roots (negative residual values in Fig. 5). An additional analysis shows that O2 in the floodwater exhibits a positive relationship with cumulative light during the day (Supporting Information Fig. S1). As pO2 in the floodwater increases during the day, a higher proportion of photosynthetically produced O2 diffuses into the root because of the shallower gradient between leaves and floodwater, and the applied Jassby & Platt (1976) model underestimates the root pO2 (positive residuals in Fig. 5). Overall, however, the residuals (vertical distances between model output and actual data point in Fig. 5) show a linear relationship with floodwater pO2, enabling us to conclude that 46% of the remaining unexplained 30% of the variation (i.e. 14%) can be attributed to variation in floodwater pO2. Thus, much of the scatter in Fig. 5 is caused by the changing pO2 in the floodwater during the course of the day and, in total, we are able to explain 84% of the observed changes in root pO2 by changes in light (70%) and floodwater pO2 (14%). These conclusions are supported by the laboratory experiments of Waters et al. (1989), where a direct influence of floodwater O2 on root surface O2 (roots in an O2-free agar solution) of submerged rice seedlings at the same light level was found; root surface O2 was lower at 10 than at 21 kPa O2 in the submergence water. In conclusion, as floodwater pO2 rises throughout the day, shoot pO2 presumably remains higher, and more of the photosynthetically produced O2 will move into the roots.
The light saturation curve in Fig. 5 shows that root pO2 no longer increases when light increases above c. 500 W m−2, indicating that underwater photosynthesis is no longer limited by light. We measured the light extinction coefficient of the floodwater to be −1.75 m−1, meaning that 65% of the incoming light is still present at mid-canopy height (depth of 25 cm). It is likely that CO2 availability under high light levels (c. 1000 W m−2 surface radiation at noon, Fig. 2) limits underwater photosynthesis with ambient CO2 concentrations of c. 50–100 μM around noon. Figure 3 shows that even leaf segments with intact gas films are CO2 limited up to c. 1000 μM CO2 at high light (PAR of 760 μmol photons m−2 s−1). Thus, CO2 would be limiting during most of the day, perhaps with the exception of the early morning (Figs 2, 3). Interestingly, both Waters et al. (1989) and Colmer & Pedersen (2008a) found that pO2 in roots and leaf sheaths, respectively, of submerged rice showed an initial peak just after the lights were turned on. In the present study, we did not see this initial peak of pO2 in the roots or sheaths, probably because of the gradual increase in light in field conditions as opposed to the sudden onset of high illumination in the aforementioned laboratory studies. The initial peaks in the laboratory studies have been suggested to result from high initial underwater photosynthesis because of buildup in the submerged tissues of respiratory CO2 during the dark period.
In our second submergence experiment, the O2 microelectrodes were inserted into the basal part of the leaf sheaths that were buried 5 cm into the soil. Here, leaf sheath pO2 reached 20–40 kPa (Fig. S2), and the fact that adventitious root pO2 never exceeded 14 kPa (Fig. 2) indicates a substantial loss of O2 from the sheaths before entry into the roots. Much of this loss could be by ROL from the basal parts of the achlorophyllous sheaths (Pedersen et al., 2011), and we observed prominent iron plaques on the parts of the sheaths that were buried in the anoxic soil.
Rice forms a gas film on the superhydrophobic leaf surfaces when immersed into water. Pedersen et al. (2009) showed that, during 7 d of submergence (12 h light : 12 h darkness, PAR of 500–600 μmol m−2 s−1, CO2 concentration of 200 μM), plants with intact leaf gas films grew as much as controls in air. By contrast, submerged plants in which the gas films had been experimentally removed survived but did not grow at all during the time of submergence. Leaf gas films enhance underwater photosynthesis (Fig. 3; and also Colmer & Pedersen, 2008b; Pedersen et al., 2009; Winkel et al., 2011) and tissue sugar status whilst submerged (Pedersen et al., 2009). In the present study, intact leaf gas films resulted in higher daytime root pO2 on the first day of submergence and, after 3 and 5 d of submergence, plants with intact leaf gas films also showed significantly higher levels of soluble sugars in the leaves (Fig. 6) relative to plants in which the gas films had been experimentally removed. On the second day of submergence, the difference in root pO2 was insignificant, suggesting that leaf gas films had either been restored or that emerging new leaves with intact gas films were able to distort any difference between control plants and plants with gas films removed (Pedersen et al. (2009) brushed new leaves that emerged, as these possessed gas films). Unfortunately, only one trace of night-time root pO2 was obtained for plants without leaf gas films (the microelectrodes were broken in other plants and/or the electrodes were misaligned and behaved erratically because of stress on the thin glass tip, resulting in rearrangement of the cathodes inside them), but this trace showed low root pO2 compared with the traces from plants with intact gas films (Fig. 2). The importance of leaf gas films for internal aeration during the night has been demonstrated previously in situ for Spartina anglica (Winkel et al., 2011) and in laboratory experiments for rice (Pedersen et al., 2009). The mechanistic functioning of the leaf gas films has not yet been fully uncovered, but it is hypothesized that the gas films act as a ‘physical gill’, a principle resembling that of plastrons in O2 uptake in some aquatic insects (Pedersen & Colmer, 2012). The importance of leaf gas films for CO2 uptake by submerged rice is demonstrated by the difference in the initial slopes of the CO2 response curves of underwater net photosynthesis (Fig. 3); removal of gas films increased by c. three-fold the effective resistance for CO2 uptake.
Little is known about the general importance of light for the survival of terrestrial plants during submergence. The few existing studies suggest a strong effect, where even low light levels can result in two- to four-fold better survival, measured as the median lethal time (LT50) (Mommer et al., 2006; Vashisht et al., 2011), consistent with underwater photosynthesis during submergence improving either internal aeration or carbohydrate status, or both. Previous work on submergence tolerance in rice seems to have focused on the importance of limited elongation to conserve carbohydrates as the main mechanism of submergence tolerance (Setter & Laureles, 1996; Das et al., 2005). However, the present study indicates that underwater photosynthesis could also be important, a hypothesis supported by observations that blocking of the early ethylene-induced chlorophyll degradation rescues the phenotype and improves survival (Ella et al., 2003). Moreover, survival correlates better with carbohydrate status at the time of de-submergence than with carbohydrate status at the time of submergence (Das et al., 2005), suggesting that continuous carbohydrate production when submerged is also of crucial advantage to submerged rice. An inherent part of the capacity for underwater photosynthesis in many terrestrial wetland plants is functioning leaf gas films (Colmer & Pedersen, 2008b; Pedersen et al., 2009; Winkel et al., 2011; Colmer et al., 2011), and we propose that the persistence of these gas films during submergence should contribute to the submergence tolerance of rice and of other wetland plants.
We thank Evangelina S. Ella, Anja Fløytrup, Melencio Apostol, James Egdane and Vichelle Dastas for their technical assistance setting up the trials and collecting and analyzing the samples for sugars and chlorophyll. Kaj Sand-Jensen and Jens Borum are thanked for valuable discussions. This work was funded by a University of Western Australia International Postgraduate Research Scholarship to A.W., the Danish Council for Independent Research grant no. 09-072482, The Crawford Fund and the International Rice Research Institute. We thank Unisense A/S for the use of equipment.