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- Materials and Methods
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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.
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- Materials and Methods
- Supporting Information
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.