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A perspective on underwater photosynthesis in submerged terrestrial wetland plants

Submergence inhibits photosynthesis by terrestrial wetland plants, but less so in species that possess leaf gas films when submerged. Floodwaters are often supersaturated with dissolved CO2 enabling photosynthesis by submerged terrestrial plants, although rates remain well-below those in air. This important adaptation that enhances survival in submerged conditions is reviewed.


Introduction
Emergent wetland plants are well adapted to waterlogged soils, but can also experience episodes of complete submergence. Complete submergence has an impact on wild species in coastal marshes and river floodplains (Armstrong et al. 1985), and many rice crops are grown in regions threatened by floods, causing submergence (Jackson and Ram 2003).
Complete submergence impedes the exchange of O 2 and CO 2 between leaves and the environment (Mommer and Visser 2005;Voesenek et al. 2006). Light availability to submerged plants also decreases, and markedly so when floodwaters are turbid (Mommer and Visser 2005;Voesenek et al. 2006). Restricted photosynthesis, but ongoing substrate consumption in respiration or fermentation, causes sugars to become depleted in submerged plants, which in turn can result in damage or even death from substrate exhaustion (Bailey-Serres and Voesenek 2008;Colmer and Voesenek 2009).
The interface between land and water is not well defined as water tables fluctuate with precipitation and evaporation, so that plants experience variable periods and depths of flooding (Sculthorpe 1967). Plants exploit niches across these dynamic flooding gradients, but functional classification of plant types lacks sharp boundaries owing to the continuum of diversity. Notwithstanding these difficulties, plants from the wettest end of the gradient have been classified into two main groups: (i) aquatic plants that primarily live completely submerged and (ii) amphibious plants that live with emergent shoots or develop water forms when submerged (Iversen 1936; cited by Sculthorpe 1967). Emergent wetland plants typically maintain a large portion of their shoots in air, but occasionally become completely submerged. To clearly distinguish these emergent plants from other wetland species with shoot portions in air (e.g. emergent amphibious plants), we refer to this functional group as 'terrestrial wetland plants' (present review;Colmer and Pedersen 2008;Pedersen et al. 2010). Such distinction is important as terrestrial wetland plants typically grow vigorously in waterlogged soils and/or areas with shallow standing water, with the depth limit being determined by capacity for transport of atmospheric O 2 to belowground tissues (Sorrell et al. 2000).
For submerged terrestrial plants, O 2 deficiency and escape responses via shoot elongation have been elucidated, revealing sophisticated signalling, changes in gene expression and altered metabolism during submergence (e.g. reviewed by Voesenek 2008, 2010). The capacity for some net photosynthesis (P N ) to continue when under water enhances plant tolerance of submergence, as P N provides O 2 for internal aeration and sugars for energy metabolism and growth (Mommer and Visser 2005).
The present review examines limitations to underwater P N by terrestrial wetland plants and compares their functioning with aquatic plants. Our focus here on underwater P N as related to the ecophysiology of submergence tolerance adds to the vast knowledge on root adaptations in wetland species. Roots of wetland plants typically contain large volumes of aerenchyma, often a barrier to radial O 2 loss, and the ability to tolerate tissue O 2 deficits and reduced phytotoxins in waterlogged soils (Armstrong 1979;Jackson and Armstrong 1999;Bailey-Serres and Voesenek 2008;Colmer and Voesenek 2009). Here, we show that underwater P N by submerged terrestrial wetland plants is limited by CO 2 availability even though floodwaters commonly contain dissolved CO 2 above air equilibrium, and so leaf traits influencing underwater P N are important for submergence tolerance.
The submergence environment during overland floods Floods differ in seasonal timing, duration, depth and frequency (e.g. Vervuren et al. 2003). Floodwater properties (e.g. water turbidity and dissolved CO 2 ) that influence plant functioning can also differ substantially; light and CO 2 available to submerged plants determine underwater P N and survival (Mommer and Visser 2005;Pedersen et al. 2010). Thus, the flooding regime and water properties influence plant species distributions in flood-prone areas (Armstrong et al. 1985;Voesenek et al. 2004). In this section, we discuss three types of flooding events that can affect terrestrial wetland plants: flash floods, seasonal floods and tidal flooding.
Flash floods occur when heavy rainfall causes water levels to rise rapidly for a variable period of time, especially as run-off moves to low-lying areas (Setter et al. 1987;Brammer 1990;Ram et al. 1999). Flash floods in some regions can be more likely to occur during specific seasons, but in other areas flash flooding is not season specific. Seasonal floods are caused by an increase in water flow that surpasses the capacity of rivers in a landscape to discharge the large volumes of water, resulting in overflow of banks and floodplains. The origin of the increased water flow can be seasonal precipitation and/or snow melt (Brammer 1990). Tidal flooding impacts coastal plains and estuarine marshes with depths determined by the moon's cycle (e.g. neap tides and spring tides). Tidal floods involve saline water, whereas overland floods are usually freshwater, with the exception of some inland catchments with salt lakes.
Flooding can occur with various combinations of chemical and physical properties in the water; O 2 , CO 2 , temperature, pH and light can all vary (Setter et al. 1987;Pérez-Lloréns et al. 2004). Seawater pH is well buffered as it contains HCO 3 2 (2.2 mM; Millero et al. 1998) and HCO 3 2 also buffers against severe depletion of dissolved CO 2 . In freshwater floods, HCO 3 2 and CO 2 concentrations are highly variable, but dissolved CO 2 is commonly above air equilibrium (Table 1). The high CO 2 concentrations typically result from respiration by organisms consuming labile carbon compounds (i.e. a net heterotrophic system); in addition, some water bodies receive CO 2 -enriched groundwater stream flows. By contrast, in net autotrophic systems photosynthesis depletes CO 2 and produces O 2 . So, O 2 concentrations in floodwaters can range from severely hypoxic (net heterotrophic) to well above air equilibrium (net autotrophic). Temperature during flooding events can also vary widely (e.g. 6-37 8C; Hamilton et al. 1997;Valett et al. 2005;Pedersen et al. 2011a), depending on location and season. Respiration increases at warmer temperatures, which can deplete O 2 , and O 2 concentration is further reduced owing to lower O 2 solubility in water as temperature increases. So, the imbalance between O 2 demand and supply to submerged terrestrial plants can be further exacerbated as temperature increases.
Flow rates during floods have only been reported, to our knowledge, in three papers: data are available for two flash floods and one seasonal river flood, and flows ranged from 0.002 to 0.3 m s 21 . Flow rates affect the thickness of diffusive boundary layers (DBLs) and thereby influence gas and nutrient exchanges with submerged plants Pedersen et al. 2009). So, underwater P N can increase with increasing flow velocity since the DBLs become thinner (Jones et al. 2000), but the response would plateau (cf. O 2 supply; Binzer et al. 2005) or even decline again if flows cause excessive shoot agitation (Madsen et al. 1993a).
Light regimes in floodwaters are dependent on several factors. When floodwaters contain suspended particles or dissolved coloured organic matter (e.g. tannins in Amazonian floodwaters; Parolin 2009), light availability will be reduced. Particle suspension can be highest during early stages of floods and particles often then settle; however, if particles settle on leaves these can still limit light. Waters of high nutrient availability typically support growth of microalgae, with dense populations of both biofilms and phytoplankton leading to lower light penetration to leaf surfaces (Sand-Jensen and Sondergaard 1981;Sand-Jensen and Borum 1991;Lassen et al. 1997) and consequently also shallower depth limits for plant colonization (Sand-Jensen 1990). Examples of light reductions are available for floodwaters in the rice fields of India and Thailand; the depth at which 50% light remained varied from 0.07 to 0.7 m (Setter et al. 1987;Ram et al. 1999).
How does the submerged environment experienced by terrestrial wetland plants compare with that of water bodies containing permanent aquatic vegetation? In brief, environments supporting healthy stands of submerged aquatic plants, such as the shallow sea, and areas within rivers and lakes, also share many of the above-mentioned constraints to plant growth. Light attenuation in the water column (caused by water itself, dissolved coloured organic matter, phytoplankton and other particles) determines the maximum depth of colonization by aquatic plants. Seagrasses typically grow down to 10% of the surface light (Duarte 1991), whereas the depth penetration of plants in freshwater lakes is down to ,1% and typically 5% of the surface light (Canfield et al. 1985). The lower light compensation points for the growth of deep -colonizing freshwater plants result from these having higher shoot-to-root ratios than seagrasses. The deepest-growing freshwater plants, such as species of Ceratophyllum and Utricularia, do not produce roots at all (Cook 1990). Similar to terrestrial floodwaters, dissolved inorganic carbon (DIC) in freshwater can also vary widely (e.g. from 0.02 to 5.6 mM in British lakes; Maberly and Spence 1983). Depending on pH, the above DIC concentrations may result in dissolved CO 2 levels from near or below air equilibrium (15 mM in freshwater at 208 C) to waters in streams/rivers, ponds and lakes that are typically supersaturated (Table 1); ponds can even contain up to 2000 mM CO 2 (133-fold air equilibrium). The temperature in most water bodies fluctuates significantly less than surrounding air due to the much higher specific heat capacity of water compared with air (Hutchinson 1957), but there are exceptions, such as in shallow rock pools with large diel fluctuations (Pedersen et al. 2011a). Finally, the flow velocity in aquatic environments also varies widely, as described earlier for terrestrial floods, from almost  (Ram et al. 1999;Setter et al. 1987); 326 (Hamilton et al. 1997;Hamilton et al. 1995;Richey et al. 2002;Valett et al. 2005 n.a., not available. O 2 was not measured in the water surveys conducted in 9, 10 and 11. stagnant conditions in ponds and deeper areas of lakes to very high velocities in rivers and in surf zones of the sea (2-3 m s 21 ; Vogel 1994). In fast-flowing water or in wavezones, the strap-shaped leaves typical of some aquatic plants are highly adaptive as this morphology reduces the pressure drag (Vogel 1994).
In summary, floodwaters faced by terrestrial plants invoke some common constraints of restricted gas exchange and lower light availability, but conditions (O 2 , CO 2 , light and temperature) differ between locations and times, posing variable challenges to plant functioning during submergence. Floodwater chemical and physical properties, in addition to the well-recognized importance of seasonal timing, duration, depth and frequency of floods (e.g. Vervuren et al. 2003), will influence plant growth and survival during submergence.

Net photosynthesis under water
Low CO 2 and/or low light can restrict P N by submerged plants (Sand-Jensen 1989). This review focuses on CO 2 acquisition. Aquatic species possess leaf traits to enhance DIC supply and thus rates of underwater P N . In Table 2, we compare the leaf traits of terrestrial wetland plants with those of submerged aquatic plants. Below we (i) summarize knowledge of morphological and anatomical leaf traits, and photosynthetic pathways including carbon-concentrating mechanisms (CCMs), and (ii) compare the rates of underwater P N by different types of aquatic and terrestrial wetland plants, as influenced by these leaf traits.

Leaf traits of terrestrial wetland plants and submerged aquatic plants
Leaf morphology determines boundary layer resistances to exchange of dissolved gases and ions (Madsen and Sand-Jensen 1991). Boundary layer resistance can limit the rates of CO 2 uptake and thus reduce underwater P N in submerged plants as diffusion is 10 4 -fold slower in water than in air (Vogel 1994). Morphological traits ( Table 2) that reduce the DBL resistance, by decreasing the distance to the 'leading edge' (Vogel 1994), include leaf shapes of small, dissected/lobed and/or strap-like leaves. In addition, aquatic leaves lack trichomes, thus avoiding the development of thicker boundary layers adjacent to their surfaces. Leaves of aquatic species also tend to be thin (Table 2), although there are several exceptions (e.g. isoetids; Sand-Jensen and Prahl 1982). Thin leaves have short internal diffusion path lengths, reducing the overall resistance for CO 2 to reach chloroplasts (Madsen and Sand-Jensen 1991;Maberly and Madsen 2002). One example is the lamina of Najas flexilis, which is only two cell layers (Tomlinson 1982). In cases where leaves are relatively thick, CO 2 is typically sourced from sediments (e.g. isoetids; Winkel and Borum 2009), and these leaves tend to be of high porosity to facilitate internal gas phase diffusion Pedersen et al. 1995 Table 2 Comparison of leaf traits influencing gas exchange and photosynthesis by terrestrial wetland plants when under water and by submerged aquatic plants. Modified from Sculthorpe (1967) with data from additional references as indicated by superscripts: 1 (Neinhuis and Barthlott 1997), 2 (Colmer and Pedersen 2008), 3 (Maberly and Madsen 2002 In addition to these morphological traits, leaves of aquatic species also have anatomical traits that further reduce diffusive resistances for CO 2 to reach chloroplasts (Table 2). Aquatic leaves lack, or have very reduced, cuticles. Diffusion across the cuticle is the main pathway of dissolved gas exchange as the leaves lack stomata, or if present, the stomata are non-functional . The diffusion path length to chloroplasts is also minimized by having these organelles in all epidermal cells, and in sub-epidermal cells the chloroplasts are positioned towards the exterior (Table 2).
Submerged aquatic plants also display physiological adaptations to increase the CO 2 concentration at Rubisco, the site of carboxylation (Table 2)-these are referred to as CCMs (Maberly and Madsen 2002;Raven et al. 2008). In submerged aquatic plants, CCMs include HCO 3 2 use (Prins and Elzenga 1989), C4 (Magnin et al. 1997), C3-C4 intermediates (Keeley 1999) and Crassulacean Acid Metabolism (CAM) photosynthesis (Keeley 1998). Carbon-concentrating mechanisms increase P N in CO 2limited submerged environments, and have also been suggested to diminish photorespiration (Maberly and Madsen 2002). Photorespiration results from the oxygenase activity of Rubisco and is promoted by a low CO 2 :O 2 ratio (Ogren 1984), a condition common in leaves when under water (Bowes 1987). The low CO 2 availability in aquatic environments would in itself lower the CO 2 :O 2 ratio. Moreover, O 2 in submerged leaves can be high as escape is slower than production in P N ; O 2 escape is not only hampered by DBLs but also by the relatively low O 2 solublity in water; CO 2 is 28.5-fold more soluble than O 2 at 208 C (Baranenko et al. 1990). Reduced photorespiration in a submerged aquatic CAM plant has been recently demonstrated (Pedersen et al. 2011b), supporting the view that CCMs do reduce photorespiration in aquatic species. By contrast with aquatic species, leaves of terrestrial wetland plants lack most of the features described above (Table 2) and so suffer from large diffusion limitations to CO 2 supply for P N when under water, unless they possess leaf gas films (Raskin and Kende 1983;Colmer and Pedersen 2008;Pedersen et al. 2009) or produce submergence-acclimated leaves (Mommer and Visser 2005). Below, we evaluate underwater P N by leaves of terrestrial wetland plants and then consider the occurrence and functioning of leaf gas films.

Net photosynthesis of aquatic and submerged terrestrial wetland plants; leaf traits enhance CO 2 supply
The most comprehensive comparison of underwater P N by aquatic and terrestrial wetland plants is the study by Sand-Jensen et al. (1992). Thirty-five species of four life forms (terrestrial, amphibious homophyllous, amphibious heterophyllous and aquatic species) were compared (listed in Appendix 1). Inclusion of data from other studies in the present analysis was constrained by differences in techniques and conditions used for underwater P N measurements, e.g. CO 2 , temperature and light (Appendix 2).
Classifications of wetland plants into functional groups are convenient, but are also imperfect as the boundaries are not sharp (see Introduction). As examples, some terrestrial wetland species produce new leaves when submerged and these can display some acclimation to the underwater environment (Mommer et al. 2007). Similarly, homophyllous amphibious plants can also display some acclimation, e.g. thinner cuticles and modestly thinner leaves when formed under water (Nielsen 1993), but these changes are far more subtle than those displayed by heterophyllous amphibious plants. Not surprisingly, different authors have classified some species into different life forms. Here, our focus is on the comparison of underwater P N of leaves formed (i) in air by terrestrial wetland species, (ii) under water by amphibious homophyllous species, (iii) under water by amphibious heterophyllous species and (iv) under water by aquatic species.
An additional noteworthy feature of the study by Sand-Jensen et al. (1992) was documentation of dissolved CO 2 levels in lowland stream habitats. Underwater P N was measured at ambient and at elevated CO 2 concentrations, to provide rates of relevance to the field situation as well as CO 2 -saturated P N for aquatic leaf types. The level of elevated CO 2 used ( 800 mM, being 50-fold air equilibrium) would have saturated P N by the aquatic leaf types. It is uncertain whether rates were CO 2 saturated for some of the terrestrial leaf types, which can require as much as 75-fold of air equilibrium CO 2 when submerged (Colmer and Pedersen 2008).
We compare the rates on the dry mass basis (Fig. 1A  and B) used by Sand-Jensen et al. (1992) and also on a projected leaf surface area basis ( Fig. 1C and D); conversions used specific leaf area (SLA) data in the literature ( Fig. 2; Appendix 1). Data for SLA were not available for three of the aquatic and three of the terrestrial wetland species in Sand-Jensen et al. (1992), so these six were omitted from the present analysis (Appendix 1).
The overall beneficial effects of aquatic leaf traits (Table 2) for underwater P N , as well as the generally poor performance of leaves of terrestrial plants, were clearly demonstrated in Sand-Jensen et al. (1992). These authors highlighted that (i) underwater P N on a mass basis increased from terrestrial, then amphibious, to truly aquatic leaf types and (ii) Danish lowland stream waters are commonly supersaturated with CO 2 , allowing even some terrestrial species to have adequate P N for growth when submerged in these habitats.
The higher P N by aquatic leaf types per unit mass with near-ambient CO 2 concentrations ( 90 -400 mM) demonstrates the higher C-return per unit of dry mass investment by these leaf types in an underwater environment as compared with terrestrial types (Fig. 1A). When external CO 2 was supplied at an elevated level of 800 mM (Fig. 1B), underwater P N values by the aquatic and heterophyllous amphibious leaves still exceeded those of the terrestrial and homophyllous leaf types. The low rates by terrestrial leaves even with elevated CO 2 further demonstrate the large diffusion limitations for CO 2 entry that restrict underwater P N .
Expression of underwater P N rates on a surface area basis, the units typically used in terrestrial plant physiology (whereas in aquatic sciences, rates are typically expressed per unit dry mass), interestingly, removes differences between the terrestrial and aquatic leaf types, at both ambient and elevated CO 2 ( Fig. 1C and D). The order of magnitude of higher SLA (Fig. 2) of aquatic and many amphibious leaf types clearly sets an upper  (1992) as it is the most comprehensive available; addition of other data was constrained by differences in techniques and conditions used (e.g. CO 2 and temperature; Appendix 2). Terrestrial, leaves formed in air by emergent wetland plants; homophyllous, leaves formed under water by amphibious wetland plants; heterophyllous, leaves formed under water by amphibious wetland plants; aquatic, leaves formed under water by submerged aquatic plants (cf. Sculthorpe 1967). Rates on a mass basis (A and B) were converted to an area basis (C and D) using the published SLA data (Fig. 2, Appendix 1). The box-whisker plot shows the median, 10 and 90 percentiles, minimum and maximum values, and means are shown as '+'; the dot in the terrestrial column indicates an outlier. Differences amongst means of the four plant groups within each panel were tested by one-way analysis of variance and Tukey's multiple comparison tests. ** P , 0.01 and *** P , 0.001. Means with the same letter within each panel do not differ significantly at the 95% confidence interval. limit for P N on an area basis. Maximum P N , however, would rarely be achieved in most aquatic environments owing to light and CO 2 limitations (Sand-Jensen 1989; Kirk 1994) so that the lower CO 2 -saturated rates of P N on an area basis for aquatic leaves would be of little consequence for their life under water.
Comparisons of the rates of underwater P N by terrestrial wetland plant leaf types with those achieved by aquatic leaf types are informative with respect to performance when submerged (Fig. 1), but here we also consider how these rates under water compare against those in air. For the terrestrial wetland species in Fig. 1, we could only find data on P N in air for three (Carex elata, Ranunculus repens and Phragmites australis; Appendix 1); P N in air at ambient CO 2 was 12.5-17 mmol m 22 s 21 . When submerged with CO 2 at levels near ambient (but well above air equilibrium in these habitats), the mean P N under water was only 9% of that in air (cf. Fig. 1C). Thus, underwater P N is greatly reduced when terrestrial wetland species become submerged.
The analyses presented above for underwater P N by leaves of terrestrial wetland plants involved experiments in which leaves growing in air were tested under water. Several terrestrial wetland species produce new leaves when submerged, and these can display some acclimation to the underwater environment (e.g. thinner cuticles and thinner leaves; Mommer et al. 2007). Acclimated leaves have decreased resistances against CO 2 and O 2 movement across the cuticle and epidermis (Mommer and Visser 2005;Mommer et al. 2007). The best example is the several-fold reduction in cuticle resistance and thus the 69-fold higher underwater P N at an external CO 2 concentration of 250 mM by Rumex palustris (Mommer et al. 2006). Although a study of seven terrestrial wetland species established the formation of a thinner cuticle as a common response when submerged, and demonstrated enhanced underwater gas exchange, the degree of this response was not correlated with submergence tolerance among these species (Mommer et al. 2007). These anatomical, and in some cases morphological (e.g. R. palustris leaves are also more elongated), changes in submerged leaves of terrestrial species are much more subtle than the altered leaf development displayed by amphibious heterophyllous species which produce true aquatic leaf types when under water (Nielsen 1993).
In summary, P N by terrestrial wetland plants is reduced markedly when they are submerged. Leaves of terrestrial wetland plants generally lack the numerous beneficial leaf traits for underwater P N possessed by aquatic plants, although new leaves can display some acclimation (e.g. thinner cuticles and higher SLA). In addition, as discussed in the next section, some leaves of terrestrial wetland species retain a gas film when submerged, a trait that also enhances underwater P N .

Leaf gas films enhance the net photosynthesis of submerged terrestrial wetland plants
Many terrestrial wetland plants have water-repellent (i.e. hydrophobic) leaf surfaces, resulting in self-cleaning by water droplets as these run off leaves (Neinhuis and Barthlott 1997). Leaf water repellence has been assessed by measurement of water droplet contact angles with the surface (Adam 1963;Brewer and Smith 1997;Neinhuis and Barthlott 1997)-angles of 1408 or more indicate a hydrophobic surface whereas angles of 1108 or less indicate a wettable surface. Water repellence (i.e. surface hydrophobicity) is determined by the micro-and nano-structures of the surface, as well as wax crystals (Wagner et al. 2003;Bhushan and Jung 2006).
Superhydrophobic leaves retain a microlayer of gas when submerged, referred to as 'gas envelopes' (Setter et al. 1989) and/or 'leaf gas films' (Colmer and Pedersen 2008). We prefer the term 'gas film' because although leaves of some species retain a gas layer on both sides (i.e. enveloped in gas), others retain a gas layer on only one side due to differences in hydrophobicity between adaxial and abaxial surfaces (Colmer and Pedersen 2008; Winkel et al. 2011). Gas films on leaves have been observed in field situations for several terrestrial wetland species when submerged in lakes, ponds, river edges and rice fields on floodplains: rice (Setter et al. 1987); P. australis, cover of New Phytologist, Volume 177(4); Spartina anglica (Winkel et al. 2011); and own observations (A. Winkel, T. D. Colmer and O. Pedersen). Information on the persistence of gas films on leaves with time following submergence is scant; gas films remained for at least 2 weeks (i.e. evaluation was terminated at 2 weeks) on leaves of Phalaris arundinacea, P. australis and Typha latifolia (all with gas films on both sides) and Glyceria maxima (gas film on only the adaxial side) in a controlled environment (Colmer and Pedersen 2008), but for some other species gas films diminish within a few days (own unpublished data; A. Winkel, T. D. Colmer and O. Pedersen).
Gas films on submerged leaves enhance CO 2 fixation, as first demonstrated for rice (9-to 10-fold increase; Raskin and Kende 1983). The beneficial effect of leaf gas films to underwater P N has also been shown for other terrestrial wetland species; at 50 mM dissolved CO 2 , gas films increased underwater P N by 1.5-to 6-fold in leaves of four wetland species (Colmer and Pedersen 2008). Data demonstrating the beneficial effect of leaf gas films on underwater P N are shown for several species in Fig. 3. Apparent resistance to CO 2 entry, at environmentally relevant CO 2 concentrations in the submergence water, was 5-fold less in leaves with gas films compared with those with gas films removed (rice and P. australis; Pedersen et al. 2009).
Leaf gas films provide an enlarged gas -water interface to promote gas exchange with the surrounding floodwater (CO 2 uptake during light periods; O 2 uptake during dark periods) (Colmer and Pedersen 2008;Pedersen et al. 2009). In addition to the enlarged gas-water interface, leaf gas films might also enable stomata to remain open when leaves are submerged. By contrast, for leaves without gas films, stomata are hypothesized to close upon submergence (Mommer and Visser 2005), so that CO 2 and O 2 must then transverse the cuticle (Mommer et al. 2004). The beneficial effect of leaf gas films on underwater P N was not only demonstrated by the marked decreases when these were removed (Fig. 3), but also leaves with this feature had higher rates of underwater P N than leaves from species without leaf gas films (Fig. 3). Thus, leaf gas films appear to enable rates of underwater P N by terrestrial leaves similar to those achieved by submergence-acclimated leaves of terrestrial wetland plants (data and discussion in Colmer and Pedersen 2008). Terrestrial species possessing leaf gas films would benefit from enhanced underwater P N during short to medium periods of submergence, depending on persistence of the films. By contrast, for species lacking leaf gas films but that produce new acclimated leaves under water, these new leaves take several days to produce so that P N would likely be less during the initial submergence period, but continued production of acclimated leaves would benefit these species during medium to prolonged submergence.
Detailed knowledge on leaf gas films is available only for rice (one cultivar only; Pedersen et al. 2009). Measurements using O 2 microelectrode profiling determined that gas film thickness varied from ,10 to 140 mm; positional differences mainly resulted from ridges on leaves (i.e. gas films thinner at the tops of ridges, thicker between adjacent ridges). Using a 'buoyancy method' to measure gas volumes on the surfaces, and within, submerged leaves, showed that tissue porosity was 19% (v/v) and the gas volume of the films was 3.8 times more than the gas within the rice leaf. Diffusive boundary layer widths adjacent to submerged leaves with gas films were surprisingly larger than those adjacent to submerged leaves without gas films, so the . These reflect the higher temperatures in tropical rice fields (O. sativa) and the lower CO 2 in seawater that submerges Spartina marshes. Gas films were removed from leaf surfaces by brushing with 0.05% Triton X-100. Species lacking leaf gas films were also brushed with Triton X-100 and showed no, or only a slight, reduction in P N . Data from Colmer and Pedersen (2008), Pedersen et al. (2009) andWinkel et al. (2011). enlarged water -gas interface provided by the gas films would have been the major mechanism that reduced resistance to gas exchange of the leaves when under water. At dissolved CO 2 concentrations of relevance to field conditions (15 -180 mM;e.g. in Thailand, Setter et al. 1987;India, Ram et al. 1999), underwater P N was enhanced 4-to 4.9-fold by gas films on leaves of rice (Pedersen et al. 2009). Underwater P N by leaves with gas films and CO 2 at near-ambient concentrations was 22% of P N in air. When gas films were removed artificially from leaves of completely submerged rice, tissue sugar levels and growth were both reduced. Thus, leaf gas films contribute to submergence tolerance of rice by enhancing CO 2 entry for underwater P N .
The experiments by Pedersen et al. (2009) also elucidated that when rice leaves are in flowing water (15 mm s 21 ; simulating low flows such as might occur across rice fields), the gas film oscillates and the transition zone between mass flow in the bulk medium and diffusion in the boundary layer was wider, and more variable, than for leaves without a gas film. Oscillations of leaf gas films in flowing water were also noted by Barthlott et al. (2010), and they reported that specialized surface hairs on the leaves of Salvinia molesta can stabilize the gas film, even in fast-flowing water (such as in streams). The leaf surface of S. molesta possesses 'eggbeater-shaped hairs' that are hydrophobic except for the tips, a feature that enables gas film formation and retention by 'pinning' the water-air interface (Barthlott et al. 2010). The presence of this feature was suggested to prevent the formation and detachment of bubbles that otherwise could occur when in fast-flowing waters (Barthlott et al. 2010). This is a very interesting leaf surface feature, although the ecophysiological significance could be debated as S. molesta is a floating plant not typically found in fast-flowing waters; the large gas volume trapped by these specialized structures on the surface of the leaves would contribute significantly to the buoyancy of this floating plant.
In addition to enhanced CO 2 uptake for photosynthesis, leaf gas films also improve O 2 uptake during darkness from floodwaters into leaves (Colmer and Pedersen 2008;Pedersen et al. 2009). Thus, leaf gas films enhance leaf O 2 status both during the daytime and during nights, with benefits also of improved internal aeration of the entire body of submerged plants. Oxygen derived from P N during light periods, as well as O 2 entry from the floodwater into leaves when in darkness, moves internally via aerenchyma to roots of rice (Pedersen et al. 2009) and rhizomes and roots of S. anglica (Winkel et al. 2011).
In conclusion, our recent studies of leaf gas films (Colmer and Pedersen 2008;Pedersen et al. 2009;Winkel et al. 2011) have supported the hypothesis by Setter et al. (1989), who observed this feature on submerged rice in field situations in Thailand, that gas films provide 'an interface between the gas and water phases for collection of CO 2 and dispersal of O 2 during the day or collection of O 2 during the night'. This mechanism is analogous to the gas layer (plastron) on some aquatic insects that provides an enlarged gas-water interface between the tracheary system and surrounding water (Thorpe and Crisp 1949;Raven 2008;Pedersen and Colmer 2012). For terrestrial wetland species, the few data available indicate that leaf gas films enable rates of underwater P N similar to those achieved by submergence-acclimated leaves, in both cases being higher than in terrestrial air-formed leaves without these features (data and discussion in Colmer and Pedersen 2008).

Conclusions and future perspectives
Submergence can have adverse effects on terrestrial wetland plants because of restricted gas exchange and low light. Floodwaters are variable in dissolved O 2 , CO 2 , light and temperature. Few data are available on key environmental parameters in various submergence environments-yet these factors influence underwater P N , plant growth and survival. Knowledge of floodwater conditions will enhance one's understanding of plant performance during submergence and enable the design of controlled experiments that better simulate particular submergence environments.
Submergence tolerance of terrestrial wetland plants is influenced by leaf traits. Although terrestrial wetland plants generally lack the numerous beneficial leaf traits possessed by aquatic plants, the few studies available demonstrate that some terrestrial species produce new leaves with a thinner cuticle under water and others possess leaf gas films. The improved gas diffusion between leaves and floodwaters enhances underwater P N and so contributes significantly to sugar and O 2 supply of submerged plants. However, studies of leaf gas film functioning are in their infancy. Our priorities are (i) quantification of the occurrence and persistence of leaf gas films amongst a wide number of wetland species, and determination of whether this trait is related to species distributions in various flood-prone wetlands (cf. analysis of shoot elongation trait; Voesenek et al. 2004) and (ii) evaluation of whether rice, or its relatives, possesses variation in leaf gas film formation and persistence, and elucidation of the underlying genetic control of this trait using the array of resources available in rice.
More broadly, there are surprisingly few studies on P N by terrestrial wetland plants when emergent and when submerged. Also lacking are measurements of P N with time after submergence and de-submergence. Future studies should compare the performances of species from various habitats, using a range of appropriate bases of expression of P N rates (area, mass, chlorophyll and leaf N) to facilitate interdisciplinary comparisons by aquatic and terrestrial plant biologists.

Sources of information
Underwater P N (used in Fig. 1