The Plant Journal (2009) 58, 147–156
doi: 10.1111/j.1365-313X.2008.03769.x
Surviving floods: leaf gas films improve O2 and CO2 exchange,
root aeration, and growth of completely submerged rice
Ole Pedersen1,2,*, Sarah Meghan Rich1 and Timothy David Colmer1
School of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, 35 Stirling
Highway, Crawley, WA 6009, Australia, and
2
Freshwater Biological Laboratory, Institute of Biology, University of Copenhagen, Helsingørsgade 51, DK-3400 Hillerød,
Denmark
1
Received 28 October 2008; revised 21 November 2008; accepted 1 December 2008; published online 19 January 2009.
*
For correspondence (fax +45 3232 1901; e-mail [email protected]).
Summary
When completely submerged, the leaves of some species retain a surface gas film. Leaf gas films on submerged
plants have recently been termed ‘plant plastrons’, analogous with the plastrons of aquatic insects. In aquatic
insects, surface gas layers (i.e. plastrons) enlarge the gas–water interface to promote O2 uptake when under
water; however, the function of leaf gas films has rarely been considered. The present study demonstrates that
gas films on leaves of completely submerged rice facilitate entry of O2 from floodwaters when in darkness and
CO2 entry when in light. O2 microprofiles showed that the improved gas exchange was not caused by
differences in diffusive boundary layers adjacent to submerged leaves with or without gas films; instead,
reduced resistance to gas exchange was probably due to the enlarged water–gas interface (cf. aquatic insects).
When gas films were removed artificially, underwater net photosynthesis declined to only 20% of the rate with
gas films present, such that, after 7 days of complete submergence, tissue sugar levels declined, and both
shoot and root growth were reduced. Internal aeration of roots in anoxic medium, when shoots were in aerobic
floodwater in darkness or when in light, was improved considerably when leaf gas films were present. Thus,
leaf gas films contribute to the submergence tolerance of rice, in addition to those traits already recognized,
such as the shoot-elongation response, aerenchyma and metabolic adjustments to O2 deficiency and oxidative
stress.
Keywords: plastrons, diffusive boundary layers, underwater photosynthesis, internal aeration, submergence
tolerance, Oryza sativa.
Introduction
Flooding has an adverse impact on plants in many ecosystems worldwide (Jackson, 2004). Submergence tolerance
can be achieved by two contrasting strategies, the ‘quiescence response’ or the ‘escape response’; both strategies
involve a number of traits (Bailey-Serres and Voesenek,
2008). The present study demonstrates the importance of
leaf gas films in the submergence tolerance of rice; a feature,
in addition to those already identified by Bailey-Serres and
Voesenek (2008), that contributes to submergence tolerance
in some wetland plants.
Water-repellent (i.e. hydrophobic) cuticles are common in
insects and plants (Neinhuis and Barthlott, 1997; Vogel,
2006), and some of these surfaces retain a microlayer of gas
when submerged. In aquatic insects, the gas layer (plastron)
ª 2009 The Authors
Journal compilation ª 2009 Blackwell Publishing Ltd
enables continued respiration underwater because of the
enlarged water–gas interface between the tracheary system
and surrounding water (Thorpe and Crisp, 1949; Hebets and
Chapman, 2000; Vogel, 2006). Leaf gas films also improve
underwater O2 and CO2 exchange (Raskin and Kende, 1983;
Colmer and Pedersen, 2008a), and have recently been
termed ‘plant plastrons’ (Raven, 2008). In plants, gas films
on submerged leaves also enlarge the water–gas interface
(cf. plastrons of insects, Hebets and Chapman, 2000), which
otherwise would be restricted to localized areas adjacent to
stomata. Moreover, as stomata are hypothesized to close
upon submergence of leaves without gas films (Mommer
and Visser, 2005), if gas films enable stomata to remain
open, at least during light periods, uptake of CO2 via stomata
147
148 Ole Pedersen et al.
(suggested by Colmer and Pedersen, 2008a) would bypass
the cuticle resistance that is a major impediment to CO2
entry in submerged leaves that do not possess gas films
(Mommer et al., 2004). Improved gas exchange by submerged leaves can enhance CO2 uptake for underwater
photosynthesis during light periods and O2 entry during
dark periods, with benefits to whole-plant internal aeration
(Sand-Jensen et al., 2005; Pedersen et al., 2006).
Gas films increased underwater net photosynthesis by
1.5–6-fold in leaves of four wetland species (at 50 mmol m)3
CO2; Colmer and Pedersen, 2008a). In rice, relative 14C
incorporation was 9–10-fold greater in leaves with gas films
present than when these were removed (Raskin and Kende,
1983). Underwater photosynthesis can enhance the tolerance of plants to complete submergence (Mommer and
Visser, 2005), as it provides O2 for internal aeration (Waters
et al., 1989; Colmer and Pedersen, 2008b) and photosynthates (Setter et al., 1989) for respiration/fermentation and
growth. The rice cultivars that are most tolerant of complete
submergence during flash floods maintain higher tissue
sugar concentrations than less tolerant cultivars, and this is
attributed to a combination of higher initial carbohydrate
concentrations and slower consumption rates during submergence (Ram et al., 2002; Jackson and Ram, 2003); the
slower sugar consumption largely results from lack of a
shoot-elongation response (Setter and Laureles, 1996; Jackson and Ram, 2003; Xu et al., 2006). In addition to differences
in sugar consumption, underwater photosynthesis can also
influence the survival of submerged rice (Setter et al., 1989;
Ram et al., 2002), although if the floodwaters are turbid, the
importance of underwater photosynthesis is diminished.
The present study demonstrates that gas films on leaves
of completely submerged rice greatly improve entry of O2
from floodwaters when in darkness, and of CO2 for photosynthesis when in light. Root aeration was improved both in
darkness and when shoots were in light, and tissue sugar
levels and growth of submerged rice were also improved.
These findings demonstrate the beneficial role of leaf gas
films for rice when completely submerged, such as occurs
during flash-flooding of lowland rice, a condition that is very
different to that studied previously (Raskin and Kende, 1983;
Beckett et al., 1988) with gas films along partially submerged
leaves of deepwater rice.
Results
Gas films enhance the internal O2 status of submerged
rice in darkness and in light
Gas films can facilitate O2 and CO2 exchange of completely
submerged leaves, with benefits for leaf respiration and
net photosynthesis (Colmer and Pedersen, 2008a). However, the influence on whole-plant aeration had not been
evaluated.
Rice plants were transferred into a two-chamber system,
so that the roots were in stagnant de-oxygenated 0.1%
agar and shoots were initially in air. The shoot was then
submerged in medium with 200 mmol m)3 CO2 and
236 mmol m)3 O2 (ionic composition given in Experimental procedures). The O2 level was air saturation, whereas
the CO2 level was 12.5 times the air saturation, as
floodwaters in rice-growing areas of Asia such as Thailand
(Setter et al., 1987) and India (Ram et al., 1999) are often
supersaturated with CO2. CO2 accumulates in flooded soils
(Ponnamperuma, 1984; Greenway et al., 2006), and the
higher solubility of CO2 compared with O2 results in a high
level of dissolved CO2 even when the floodwater O2 level
remains close to air saturation. When submerged, gas
films were evident along all shoot tissues, presumably
because the surfaces are hydrophobic; the contact angles
() of water droplets placed on horizontally held leaf
blades, determined as described by Neinhuis and Barthlott
(1997), were 148 2 (adaxial) and 149 1 (abaxial). Using
O2 microelectrodes, the pO2 dynamics within adventitious
roots were measured while shoot conditions were manipulated. Recordings are shown for plants with shoots in
light (Figure 1a) and in darkness (Figure 1b), and the quasi
steady-state values for replicated experiments are given in
Table 1.
For plants with shoots in light and initially in air, root
pO2 was approximately 12 kPa (Figure 1a and Table 1); as
expected, the pO2 was lower than atmospheric partial
pressure (approximately 20.6 kPa) as O2 will have been
consumed along the diffusion path within the aerenchymatous roots (see Armstrong, 1979). Upon submergence
of the shoots in light, root pO2 increased (Figure 1a and
Table 1), presumably reflecting an increase in shoot pO2
due to impeded outward diffusion of photosynthetically
produced O2 when tissues are surrounded by water
(Colmer and Pedersen, 2008b). Removal of leaf gas films
resulted in a decline in root pO2 to just below the initial
level (Figure 1a and Table 1), as O2 production via photosynthesis declined due to impeded entry of CO2 (see
section on underwater photosynthesis). De-submergence
of the shoot resulted in a rapid return of root pO2 to near
the original levels prior to shoot submergence (Figure 1a
and Table 1), indicating that removal of leaf gas films with
Triton X-100 had no irreversible effect on shoot gas
exchange when in air.
For plants submerged in darkness (Figure 1b), root pO2
declined to about 25% of that when submerged in light
(Table 1). Removal of leaf gas films caused the root pO2 to
decline to very low levels (approximately 0.1 kPa) when
shoots were in darkness (Figure 1b and Table 1), as diffusion
of O2 from the surrounding floodwater into the leaves is
impeded. De-submergence of the shoot resulted in a rapid
increase in root pO2 to the level prior to submergence
(Figure 1b and Table 1).
ª 2009 The Authors
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 58, 147–156
Gas films contribute to rice submergence tolerance 149
(a) 16
14
Root pO2 (kPa)
Table 1 Influence of leaf gas films on pO2 in roots of rice (Oryza
sativa L.) when submerged with shoots in light or darkness
Gas films removed
Treatment
12
10
Shoot submerged
8
Shoot de-submerged
6
4
2
0
0
1
2
3
4
5
6
7
(b) 16
Root pO2 (kPa)
14
Shoot in light
12
8
Gas films removed
6
Shoot
de-submerged
Shoot in dark
4
11.8a,c 0.2
13.5b 0.5
10.5a 0.1
12.8b,c 0.2
9.9a,c 0.5
3.4d 0.8
0.1e 0.04
10.8a,b,c 0.9
Four-week-old plants were mounted in a two-compartment chamber
with the roots in deoxygenated 0.1% agar medium and the shoot in
air and then in artificial floodwater with 200 mmol m)3 CO2. An O2
microelectrode was inserted into the cortex of an adventitious root
(8–12 cm long), 20–25 mm behind the root tip. Root pO2 was recorded
with the shoot in light or in darkness (both at 30C). Values are
means SE (n = 4–7, each replicate was a different plant). Different
letters indicate significant differences (Tukey test, P < 0.05).
Shoot submerged
10
Shoot in light
Shoot in air (n = 7)
Shoot completely
submerged (n = 4)
Gas films removed, shoot
submerged (n = 4)
Shoot de-submerged (n = 4)
Shoot in darkness
Shoot in air (n = 4)
Shoot completely submerged (n = 7)
Gas films removed, shoot
submerged (n = 7)
Shoot de-submerged (n = 4)
Root pO2 (kPa)
2
0
0
1
2
3
4
Time (h)
5
6
7
Figure 1. Dynamics of O2 partial pressures (pO2) in adventitious roots of 4week-old rice (Oryza sativa L.) in light (a) or darkness (b), before and after
submergence, and with and without removal of gas films (lamina and sheath
exteriors), at 30C.
O2 microelectrodes were inserted 20–25 mm behind the tip of intact adventitious roots of plants with roots in deoxygenated 0.1% agar medium. Quasisteady state pO2 in the root was established with the shoot in air, then
submerged (200 mmol m)3 CO2), then the shoot was gently lifted out of the
water and brushed with 0.1% Triton X-100 so that gas films were absent when
re-submerged, and finally de-submerged so that the shoot was again in air.
The small peak in pO2 in the trace taken in darkness (b) during gas film
removal was due to entry of atmospheric O2 during brushing. Tissue
porosities (%) were: leaf blades, 19.2 1.2; sheaths, 30.7 2.9; adventitious
roots, 26.7 3.4. Means of replicated measurements of pO2 taken from
different plants are given in Table 1.
Gas films: volume, dimensions and diffusive
boundary layers
When submerged, O2 and CO2 exchange between leaves
and the surrounding water is impeded due to a 104-fold
slower diffusion of gases in water compared with air (Armstrong, 1979; Vogel, 2006). Diffusive boundary layers,
therefore, are a major component of the resistance to uptake
of dissolved gases, and so were quantified for O2 uptake by
leaf segments with or without gas films (Figure 2 and
Table 2).
Figure 2 shows typical examples of diffusive boundary
layers, measured using O2 micro electrode profiling, adjacent to leaf segments with (Figure 2a) or without (Figure 2b)
gas films when in darkness and with air-saturated medium
flowing across the segments fixed in a flume tank. The
diffusive boundary layers in the liquid phase (both ‘true’ and
‘effective’; see Figure 2) were thicker on leaf segments with
gas films compared to those without gas films (examples in
Figure 2; means in Table 2). Furthermore, the transition
zone between turbulent and laminar flows was also wider on
leaf segments with gas films (Figure 2 and Table 2). We
tested whether these effects could be attributed to Triton X100 per se by conducting measurements on the aquatic
leaves of Haloragis brownii (no natural gas film) with or
without pre-brushing with Triton X-100; the Triton X-100
treatment had no effect on the diffusive boundary layers
(control 118 2 lm; Triton X-100-treated 116 2 lm;
mean SE, n = 5). The results for rice leaves demonstrate
that the better aeration in plants with gas films was
not related to lower liquid-phase diffusive boundary layer
resistances.
The O2 concentration gradient within the true diffusive
boundary layer can be used to calculate net O2 fluxes. The
gradient at the position measured was larger for leaves
without gas films, implying that these had faster rates of net
O2 uptake. However, when the actual O2 consumption by
whole leaf segments was determined by depletion in wellmixed closed chambers, the rates for leaf segments with or
without gas films at atmospheric saturation did not differ
(pooled mean = 0.6 lmol m)2 sec)1, Table 2). The net fluxes
calculated from the O2 profiling in the flume system for leaf
segments without gas films were also 0.6 lmol m)2 sec)1,
whereas for those with gas films it was three times lower
ª 2009 The Authors
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 58, 147–156
150 Ole Pedersen et al.
Table 2 Leaf gas film volume and dimensions, surface pO2, O2
consumption rates, leaf porosity, and boundary layer condition of
rice (Oryza sativa L.) leaf segments with or without gas films at 30C
900
800
700
600
500
400
300
200
100
0
13
With gas films
Transition zone
Distance (µm)
(a) 1000
Effective DBL = 430 µm
True DBL = 160 µm
Leaf surface
Gas film = 90 µm
14
15
16
17
18
19
20
21
22
900
800
700
600
500
400
300
200
100
0
13
Without gas films
Transition zone
Distance (µm)
(b) 1000
Effective DBL = 175 µm
Leaf surface
14
15
16
17 18 19
pO2 (kPa)
20
21
22
Figure 2. Examples of diffusive boundary layers adjacent to completely
submerged leaf segments of rice (Oryza sativa L.): lamina segments 60 mm
long and 8–10 mm wide either with gas films (a) or without gas films (0.1%
Triton X-100-treated) (b), in darkness in a flume with mean bulk flow velocity
of 15 mm sec)1, at 30.
Profiles were measured using an O2 microelectrode (tip diameter 10 lm).
Dimensions of the true, and effective, diffusive boundary layers were
estimated according to Jørgensen and Revsbech (1985), and are indicated
in (a) but not in (b) as the extension of the two zones is roughly the same in the
example shown in (b). Means of replicate values taken from leaf segments
from different plants are given in Table 2.
(Table 2). We hypothesize that the discrepancy between the
two techniques for leaves with gas films could result from
position effects. With gas films present, longitudinal O2
diffusion would be rapid, so if O2 uptake was enhanced at
the leading edge of the leaf segment, due to eroded diffusive
boundary layers at that edge, then uptake at the position
measured (midpoint of the 60 mm leaf segment) would be
lowered, as O2 supply to tissue at this point would also be
via longitudinal O2 diffusion within the gas layer. This
possibility is supported, assuming equal tissue O2 demands
within the two treatments (cf. respiration chamber results),
by the finding of a higher pO2 of 18.2 kPa at the surface of
leaves with gas films, compared with 13.9 kPa on those
without (Table 2).
The O2 profiles were also used to estimate the thickness of
gas films on the adaxial side of leaf segments, as high
diffusivity in air means O2 concentration gradients are not
Parameter
True diffusive boundary
layer (lm) (P < 0.05)
With gas film
Without gas film
Effective diffusive boundary
layer (lm) (P < 0.05)
With gas film
Without gas film
Transition zone between turbulent flow
and viscous laminar flow (lm) (P < 0.05)
With gas film
Without gas film
Leaf surface pO2 in darkness (kPa) (P < 0.05)
With gas films
Without gas films
Leaf O2 consumption (lmol O2 m)2 sec)1)
calculated from O2 profiles (P < 0.05)
With gas films
Without gas films
Leaf O2 consumption (lmol O2 m)2 sec)1)
determined using respiration
chambers (P > 0.05)
With gas films
Without gas films
Leaf gas film thickness (lm) (P > 0.05)
determined by the buoyancy method
Adaxial
Abaxial
Average
Leaf gas film thickness range (lm)
Adaxial
determined by the microelectrode
method
Leaf porosity (% gas volume per
unit tissue volume)
Gas film volume relative to internal
leaf gas volume
Mean SE (n)
266 38 (6)
124 16 (4)
360 35 (6)
156 17 (4)
373 53 (6)
198 42 (4)
18.2 0.1 (6)
13.9 0.1 (4)
0.21 0.01 (6)
0.60 0.21 (4)
0.55 0.01 (8)
0.63 0.04 (8)
67 4 (4)
57 12 (4)
62 4 (8)
<10–140 (6)
19.2 1.2 (4)
3.8 0.4 (4)
Values are means SE with the number of replicates in parentheses
(each replicate comprised a leaf segment from different plants). For
explanations of true and effective diffusive boundary layers, and the
transition zone, see the legend to Figure 2. P values refer to
comparisons between with or without gas films, within each
parameter measured.
established over short distances (see vertical portion of
the profile immediately adjacent to the leaf surface in
Figure 2a), whereas the diffusive boundary layer profiles
were clearly evident in water (Figure 2a,b). Gas film thickness varied from <10 to 140 lm (mean 89 23 lm; Table 2),
a mean value that is not significantly different from that
obtained using a buoyancy method, on segments before
and after gas film removal, to determine gas film volumes
per unit area, from which the mean gas film thickness was
calculated (62 4 lm; Table 2). The leaf blade thickness
was 156 16 lm (mean SE, n = 12), and so the gas film
volume (adaxial plus abaxial sides) was roughly equal to the
ª 2009 The Authors
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 58, 147–156
Gas films contribute to rice submergence tolerance 151
tissue volume (Table 2). Consequently, the volume of gas
within the films was almost four times higher than the
internal leaf tissue gas volume (Table 2).
Gas films enhance underwater net photosynthesis, tissue
sugar levels and growth of completely submerged rice
The experiments described above established that leaf gas
films enhance the internal O2 status of submerged rice. In
the experiments described in this section, we evaluated
whether gas films are also beneficial to underwater net
photosynthesis, tissue sugar status, and ultimately plant
growth when completely submerged.
Gas films enhanced underwater net photosynthesis in
leaf segments of rice across a wide range of external CO2
concentrations (Figure 3). At low-to-medium CO2 availability (15–180 mmol m)3), underwater net photosynthesis was
4–4.9-fold higher in leaf segments with gas films, whereas
the rates in leaf segments with or without gas films were
equal at high CO2 supply (2000 mmol m)3) (Figure 3). The
low-to-medium CO2 concentrations are of relevance to
some field conditions such as in Thailand (Setter et al.,
1987) or India (Ram et al., 1999), whereas the high CO2
levels were used to investigate whether the rates for leaf
segments with or without gas films converge. The apparent
resistance to CO2 entry was five times less in leaf
segments with gas films (legend to of Figure 3), such that
5
Net photosynthesis
(µmol m–2 sec–1)
With gas films
4
3
2
Without gas films
1
0
0
500
1000
1500
2000
CO2 concentration (mmol m–3)
Figure 3. Underwater net photosynthesis in leaf segments from 4-week-old
rice (Oryza sativa L.) either with gas films or without gas films (0.1% Triton X100-treated), as a function of CO2 in the medium. Lamina segments (30 mm)
were incubated in glass cuvettes attached to a rotating wheel within a water
bath at 30C, and net photosynthesis was measured as O2 evolution over 60–
90 min. The data were fitted to the equation of Jassby and Platt (1976), with
Pmax estimated to be 4.00 0.08 lmol m)2 sec)1 (r2 = 0.93) with gas films
and 4.66 0.30 lmol m)2 sec)1 (r2 = 0.97) without gas films, the two means
not being significantly different. The apparent resistance to CO2 uptake was
fivefold lower with gas films present (57 850 sec m)1) than without
(274 375 sec m)1). The apparent resistance to CO2 uptake was determined
as 1 divided by the slope of a linear regression for the 15–180 mmol m)3
range. Values are means SE (n = 5, each replicate being a leaf segment
from a different plant).
underwater net photosynthesis was enhanced at lower CO2
availability. By increasing the external CO2 supply,
however, the higher apparent resistance to CO2 entry in
leaf segments without gas films could be overcome
(Figure 3). Nevertheless, the maximum rate of 4 lmol m)2 sec)1 under water was below that of leaves in air
(11.3 0.3 lmol m)2 sec)1 with 380 ll L)1 CO2 and
17.8 0.7 lmol m)2 sec)1 with 760 ll L)1 CO2; PAR
350 lmol m)2 sec)1; mean SE, n = 4).
The greater rates of underwater net photosynthesis by
leaves with gas films (Figure 3) not only enhanced the
internal O2 status of completely submerged rice (Figure 1
and Table 1), but also enhanced tissue sugar concentrations
(Figure 4a) and growth (Figure 4c,d). After 7 days of complete submergence, the total sugar levels in leaves of plants
without gas films had declined to 66% of the level in those
with gas films; moreover, total sugar levels in submerged
plants with gas films did not differ from levels when the
shoot was in air (Figure 4a). In roots, however, although
total sugar levels in plants without gas films were 63% of
those with gas films (Figure 4b), the variation was large and
so this difference was not significant at the 5% level. The
growth of submerged plants without gas films was
impeded; the shoot dry mass was 49% of the values for
submerged plants with gas films, and the root dry mass was
33%. The beneficial effect of gas films during complete
submergence is further demonstrated by the submerged
plants with gas films not differing with respect to shoot
growth from the controls with shoots in air (Figure 4c),
although root growth was reduced in comparison with
controls (Figure 4d).
For plants with shoots in air, those treated with Triton X100 showed a decline in root growth (Figure 4d), but not in
shoot growth (Figure 4c). A second experiment established
that net photosynthesis in air following Triton X-100
treatment and rinsing with submergence medium was
initially depressed to 60% of that in untreated plants, and
the rate recovered by 60 min (data not shown). Plants
without Triton X-100 treatment were also rinsed; the
solution was instantly shed from these hydrophobic leaf
surfaces, and so net photosynthesis in these plants did not
differ before and after rinsing (data not shown). The
inhibitory effect of surface wetness on leaf gas exchange,
as probably occurred in the Triton X-100-treated plants, has
also been reported for terrestrial species with naturally low
leaf water repellency (Smith and McClean, 1989). Thus, a
transient decline in photosynthesis is the likely cause of the
lower growth in ‘shoot in air controls’ (i.e. in air with Triton
X-100 treatment) in comparison with plants in air without
Triton X-100 treatment. This effect, however, would not
have influenced the submerged plants, as Triton X-100 per
se did not influence boundary conditions when under water
(see results in preceding section for the check of Triton X100 effects on aquatic leaves of Haloragis brownii).
ª 2009 The Authors
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 58, 147–156
a
ab
b
400
c
200
0
0.15
This study demonstrates that leaf gas films facilitate
underwater net photosynthesis, with resultant improvements in tissue pO2, sugar levels and growth of rice when
completely submerged. Enhanced growth during submergence, due to leaf gas films, was evident for both shoots
and roots (Figure 4), and the higher pO2 in roots (Figure 1
and Table 1) would also have promoted radial O2 loss from
root tips, which is of importance for growth into anaerobic
substrates (see Armstrong, 1979; Colmer, 2003). The beneficial effect of gas films on submerged leaves (present study)
reinforces the view that underwater photosynthesis is a key
determinant of the survival and growth of plants during
complete submergence (Mommer and Visser, 2005; BaileySerres and Voesenek, 2008).
Underwater photosynthesis is a major source of O2 for
submerged wetland plants (e.g. rice; Colmer and Pedersen,
2008b; Waters et al., 1989), and also provides sugars. For
rice that had been completely submerged for 7 days,
underwater net photosynthesis was enhanced by leaf gas
films, and this enabled maintenance of tissue sugar levels
and growth (Figure 4). The importance of underwater photosynthesis for maintenance of sugar levels and growth of
completely submerged rice has also been demonstrated by
b
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tr
s
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ai
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ti
b
oo
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oo
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as
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ou
w
b
Su
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s
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ith
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b
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ith
ai
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iti
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c
a
r
0.00
on
0.00
c
a
s
0.05
r
0.05
oo
b
0.10
ith
a
0.20
Figure 4. Total sugar levels in (a) leaves (lamina
only) and (b) roots, and dry mass of (c) shoots
and (d) roots of rice (Oryza sativa L.) with shoots
in air (with or without 0.1% Triton X-100 treatment) or when completely submerged with or
without gas films.
Fourteen-day-old rice with roots in containers of
stagnant, de-oxygenated 0.1% agar with nutrient
solution were either kept for an additional 7 days
with shoots in air within transparent Perspex
tanks (shoot in air), brushed with 0.1% Triton
X-100, rinsed and retained in air (shoot in air
control), completely submerged in a medium
with 200 mmol m)3 CO2 (sub with gas films), or
leaves and sheaths were brushed with 0.1%
Triton X-100 to prevent gas films when completely submerged (sub without gas films).
Samples were also taken when treatments were
imposed (initial). Gas films on submerged leaves
had positive effects on growth (Tukey test,
P < 0.05, indicated by different letters). Assuming exponential growth, whole plant relative
growth rates were: shoot in air = 0.184 day)1;
shoot in air control = 0.155 day)1; sub with gas
films = 0.181 day)1; sub without gas films =
0.071 day)1. Gas films on submerged leaves
had positive effects on total sugar concentration
in leaves (Tukey test, P < 0.05, indicated by
different letters), but differences were not significant for roots. Values are means SE (n = 4,
each replicate being a different plant).
Su
0.15
Sh
b
0.25
ai
ac
Sh
b
0
rc
0.20
0.10
b
0.30
bc
0.25
b
200
Su
0.30
a
400
0.35
(d)
b
b
600
n
b
800
ti
600
(c) 0.35
Shoot dry mass (g)
Root sugar concentration
(µmol hexose equiv. g–1 dry mass)
(b)
800
fil
(a)
Root dry mass (g)
Leaf sugar concentration
(µmol hexose equiv. g–1 dry mass)
152 Ole Pedersen et al.
CO2 enrichment of the submergence water (Setter et al.,
1989). The present finding of the benefit of leaf gas films for
the whole-plant carbon balance of submerged rice extends
an earlier prediction of improved leaf-level carbon balance
of submerged Phragmites australis when gas films were
present (Colmer and Pedersen, 2008a) and an earlier observation of enhanced 14CO2 uptake by submerged rice leaf
segments with gas films present (Raskin and Kende, 1983).
Even with gas films present, however, the maximum rate of
net photosynthesis under water was approximately 40% of
that in air with ambient CO2, and 22% of that when high CO2
levels were supplied in air. Nevertheless, underwater photosynthesis facilitated by leaf gas films (present study), as
well as the consumption of sugars during submergence
(Ram et al., 2002; Jackson and Ram, 2003; Bailey-Serres and
Voesenek, 2008), could both contribute to the carbon
balance of rice when completely submerged, and so determine survival. In turbid waters, however, low light would
restrict photosynthesis.
The proposed mechanisms by which gas films on
submerged leaves facilitate underwater net photosynthesis
include the larger gas–water interface for CO2 uptake from
the floodwater than if restricted to localized areas adjacent to
stomata (cf. plastrons of insects, Hebets and Chapman,
2000), and the possibility that stomata may remain open for
ª 2009 The Authors
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 58, 147–156
Gas films contribute to rice submergence tolerance 153
gas movements (suggested by Colmer and Pedersen,
2008a). Although stomata are hypothesized to close upon
submergence of leaves without gas films (Mommer and
Visser, 2005), data on this subject are lacking, and reliable
measurements to compare leaves with or without gas films
are not available, so the suggestion by Colmer and Pedersen
(2008a) remains speculative. Nevertheless, the present study
clearly demonstrates that apparent resistance to CO2 entry at
environmentally relevant concentrations was fivefold less in
leaves of rice with gas films compared to those without.
Similarly, the presence of gas films on leaves of Phragmites
australis also reduced the apparent resistance to CO2 uptake
5.2-fold (calculated from Figure 2 in Colmer and Pedersen,
2008a). In the case of rice, the higher apparent resistance to
CO2 entry in leaves without gas films was, as expected,
overcome by increasing the external CO2 supply (Figure 3),
whereas, unexpectedly, this was not the case for Phragmites
australis (Figure 2 of Colmer and Pedersen, 2008a). The
enhanced underwater gas exchange would also promote
outward O2 diffusion during light periods, so that the level of
photorespiration would presumably also be lower; photorespiration can be substantial in submerged rice (Setter
et al., 1989).
Diffusive boundary layers in the liquid phase remain a
major resistance component to dissolved gas exchange
between the floodwater and leaves even when gas films are
present, as these layers were thicker for leaf segments with
gas films than for those without (Figure 2 and Table 2). We
speculate that the elastic properties of the gas films, which
fluctuate between a compressed and extended state as highdensity packets of water pass along the leaves, would
dissipate energy and so decrease erosion of the diffusive
boundary layer (Vogel, 2006). The fluctuating gas layer
would also have resulted in a wider transition zone between
laminar and turbulent flows adjacent to the leaf segments
when in flowing water (Figure 2). Despite the thicker diffusive boundary layers (determined from O2 profiles; Figure 2), the leaves with gas films had enhanced CO2 uptake
(determined from underwater photosynthesis; Figure 3), so
decreases in other resistance components (see above)
outweighed the change in diffusive boundary layers. Understanding of gas transport processes across water–air interfaces in natural environments is an active area of study
(Banerjee, 2007); knowledge from interface transport (Turney and Banerjee, 2008) as well as bubble gas-exchange
modeling (Woolf et al., 2007) should both aid understanding
of the physics of leaf gas films, although the gas films would
be considered ‘stationary bubbles’ and are not spherical.
In summary, leaf gas films make a substantial contribution to the submergence tolerance of rice; internal aeration,
sugar status and growth were all enhanced by the films.
Whether leaf gas films differ amongst rice cultivars should
be evaluated. Some other wetland plants also possess gas
films, with benefits for underwater net photosynthesis
(Colmer and Pedersen, 2008a), and some species that lack
gas films can acclimatize so that newly produced leaves
have improved gas exchange underwater (e.g. thin cuticles,
thin leaves, chloroplast re-orientation; Mommer et al., 2004,
2005, 2007). In the wider context of submergence tolerance
in plants, as reviewed recently by Bailey-Serres and Voesenek (2008), leaf gas films and associated higher rates of
underwater net photosynthesis, and thus sugar and O2
supply, would be beneficial for species/cultivars with a shoot
elongation ‘escape‘ response or a non-elongation ‘quiescence’ response.
Experimental procedures
Plant material
Oryza sativa L. var. Amaroo was raised in aerated nutrient solution
(Colmer and Pedersen, 2008b), and then pre-treated for the final
7 days in stagnant 0.1% w/v agar nutrient solution, with shoots
remaining in air, prior to use in experiments. The ages of plants
used and the numbers of replicates (different plants, or tissues from
different individuals), in each experiment, are given in of the table
and figure legends.
Morphological, chemical and biophysical properties
of leaves
The most recent fully expanded leaves were characterized for
selected properties. Water repellency of leaf surfaces was assessed
by measuring the contact angle of a 5 mm3 droplet of water on the
leaf surface (Adam, 1963; Brewer and Smith, 1997; Neinhuis and
Barthlott, 1997) as described by Colmer and Pedersen (2008a). Leaf
thickness was measured on transverse sections of lamina, using a
microscope equipped with a digital camera.
Surface gas film volume and tissue porosity (% gas volume per
unit tissue volume) were measured by determining tissue buoyancy
before and after vacuum infiltration of the gas spaces with water
(Raskin and Kende, 1983), using equations as modified by Thomson
et al. (1990). Triton X-100 (0.1% v/v) was used to remove surface gas
films on the tissues.
Diffusive boundary layers adjacent to submerged
leaf segments
For each replicate, two lamina segments of approximately 60 mm
length and 8–10 mm width were taken halfway up the blade of the
most recently fully expanded leaf. One was used as a control (with
gas films) and the other was used for gas film removal treatment.
Segments used for gas film removal treatment were brushed on both
sides with a fine paintbrush soaked in 0.1% v/v Triton X-100 in
incubation medium (composition given below in the section on
underwater net photosynthesis), and then washed for 5 sec, three
times, in fresh incubation medium. This treatment prevented formation of gas films on the lamina surfaces when submerged (Raskin
and Kende, 1983), and did not cause any ‘flooding’ of internal gas
spaces (Colmer and Pedersen, 2008a). The control segments (with
gas films) were also washed in incubation medium prior to use.
Lamina segments were mounted on double-sided adhesive tape
in a custom-built flume tank with a mean bulk flow velocity of
15 mm sec)1 parallel to leaf segments. The flume allowed O2
ª 2009 The Authors
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 58, 147–156
154 Ole Pedersen et al.
microeletrodes (tip diameter = 10 lm, OX-10, Unisense A/S, http://
www.unisence.com) to penetrate the lamina segments from below,
with the tip eventually protruding into the air-saturated incubation
medium flowing above the segments. Thus, diffusive boundary
layers were measured without the microelectrode itself affecting
flows, which would not have been the case if the electrode had been
moved towards the leaf surface from above instead of from within
and away (see Glud et al., 1994). The O2 microelectrodes were
connected to a pA meter (PA2000, Unisense A/S), and electrodes
were advanced in steps of 10 lm every 5 sec using micromanipulators (MM33, Unisense A/S). All measurements were performed in
darkness at 30C. Diffusive boundary layer thickness was calculated
from the O2 gradients, as described by Jørgensen and Revsbech
(1985).
Dark respiration by leaf segments
Dark respiration of lamina segments of approximately 30 mm
length was measured in a microrespiration system. Measurements
were taken using 4 ml glass chambers with a capillary hole in the
glass stopper (MR Ch-4000, Unisense A/S), through which an O2
microelectrode (OX-MR, Unisense A/S) was inserted. The medium
used was identical to that used for measurement of underwater net
photosynthesis (see below), with a CO2 concentration of
50 mmol m)3 at 30C (for further details, see Colmer and Pedersen,
2008a).
Underwater net photosynthesis by leaf segments
For each replicate leaf, two lamina segments of approximately
30 mm length were taken halfway up the blade of the most recently
fully expanded leaf. One was used as a control (with gas films) and
another was used as the treatment in which gas films were removed
by brushing with 0.1% v/v Triton X-100 in incubation medium, and
then washed in incubation medium without Triton X-100. Underwater net photosynthesis by the lamina segments was measured
using the method described by Colmer and Pedersen (2008a).
Measurements commenced within 15 min of excision. Glass
cuvettes (35 ml) with stoppers contained individual lamina
segments in incubation medium and two glass beads for mixing as
the cuvettes rotated on a wheel within an illuminated water bath at
30C.
Photosynthetically
active
radiation
(PAR)
was
350 lmol m)2 sec)1, measured inside the submerged glass
cuvettes (using a 4p US-SQS/L quantum sensor, Walz, http://www.
walz.com).
The incubation medium contained 0.50 mol m)3 Ca2+,
0.25 mol m)3 Mg2+, 1.00 mol m)3 Cl), 0.25 mol m)3 SO42) and
5.0 mol m)3 MES, with pH adjusted to 6.00 using KOH. The
dissolved O2 concentration in the incubation medium was set at
50% of air saturation by bubbling in a 1:1 ratio (by volume) of N2 and
air (prior to adjustment of dissolved CO2); this avoided build-up of
high O2 levels during the measurements that might have led to
photorespiration and thus decreased net photosynthesis, as previously described for submerged rice leaves (Setter et al., 1989). As
flasks were incubated in light immediately after adding the lamina
segments, and as the segments produce O2 when in light, there was
no risk of tissue hypoxia. Dissolved CO2 treatments were imposed
by adding specific concentrations of KHCO3 to the incubation
medium, with pH always adjusted to pH 6.00 using various amounts
of KOH depending on the amount of KHCO3 added, to provide a
range of CO2 concentrations from 15 (air saturation) to
2000 mmol m)3 (Stumm and Morgan, 1996). K2SO4 was added as
required in the various treatments so that the K+ concentration was
equal across treatments.
Following incubations of known duration (60–90 min), dissolved
O2 concentrations in the bottles were measured using a Clark-type
O2 mini-electrode (OX-500, Unisense A/S) connected to a pA meter
(PA2000, Unisense A/S). The electrode was calibrated immediately
before use. Dissolved O2 concentrations in bottles prepared and
incubated in the same way as described above, but without lamina
segments, served as blanks. The projected area of each lamina
segment was measured using a leaf area meter (Li-Cor LI-3000,
http://www.licor.com), and the fresh and dry masses (after freeze
drying) of each segment were determined.
Net photosynthesis in air
Net photosynthesis in air at a PAR of 350 lmol m)2 sec)1 and
ambient (380 ll L)1) and high (760 ll L)1) CO2 were measured at
30C on intact leaves (most recent fully expanded) using a flowthrough leaf cuvette connected to an infra-red gas analyser (LI-6400,
Li-Cor).
Root aeration of whole plants
Four-week-old plants were transferred into a horizontal chamber
(length 0.8 m) made from 150 mm diameter PVC pipe sliced
lengthways in half, with caps at each end and a divider fitted
300 mm from one end (Colmer and Pedersen, 2008a). The root–
shoot junction was positioned 10 mm below the divider, so that the
roots were submerged in incubation medium (see net photosynthesis measurements for composition) that also contained 0.1% w/v
agar to prevent convective movement and had been deoxygenated
by prior flushing with N2. Initially, shoots were always in air. The
root compartment was covered with glass plates and a small
opening enabled insertion of an O2 microelectrode. The shoot base
was held in place within the divider using BluTac putty (Bostik,
http://www.bostik.com). The shoot compartment was filled with
incubation medium (without agar) containing 200 mmol m)3 free
CO2 and O2 in air equilibrium (236 mmol m)3). Any possible contact
between leaves and air was prevented by covering the submergence solution with transparent polyethylene sheeting. Experiments were performed in a room kept at 30C. During light periods,
PAR (350–400 lmol m)2 sec)1) was provided by halogen spotlights.
Clark-type O2 microelectrodes with a guard cathode and tip
diameter of 25 lm (OX-25, Unisense A/S) (Revsbech, 1989) were
used. A microelectrode was inserted into the root cortex 20–25 mm
behind the root tip using a micromanipulator (MM5, Märzhäuser,
http://www.marzhauser.com). The microelectrode was connected
to a pA meter (PA8000, Unisense A/S) and the outputs were logged
every 10 sec on a computer using an analog to digital converter
(ADC-16, Pico Technology, http://www.picotech.com). O2 concentrations in the shoot submergence solution and in the 0.1% stagnant
agar solution in the root compartment were both monitored using
Clark-type O2 minielectrodes (OX-500, Unisense A/S); in all experiments, the solution in the shoot compartment remained close to air
saturation and O2 in the bulk medium of the root compartment
increased to a maximum of 0.5 kPa. The water temperature was
recorded using type-K thermocouples connected to a resistance
converter (TC-08, Pico Technology), and remained at 30 0.5C.
Growth experiment for whole plants
To investigate the influence of leaf gas films on sugar status and
growth, 14-day-old plants were completely submerged for 7 days.
The experiment was a 2 · 2 factorial design, and consisted of controls with shoots in air, a second control with shoots in air but
ª 2009 The Authors
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 58, 147–156
Gas films contribute to rice submergence tolerance 155
brushed with 0.1% Triton X-100, submerged with gas films, and
submerged and Triton X-100-treated to remove gas films on leaves
and sheaths. In all treatments, shoots were also rinsed with incubation medium. The experiment was performed in a constantenvironment room (30C, 12 h light/12 h darkness, 80% relative
humidity, PAR of 500–600 lmol m)2 sec)1).
All plants were placed with their roots in 500 ml darkened bottles
containing stagnant 0.1% agar nutrient solution (Colmer and
Pedersen, 2008b), and then into transparent acrylic plastic cylindrical tanks. Half of the Perspex tanks did not contain any solution;
those assigned to the submergence treatment were filled with 12 L
of incubation medium (see net photosynthesis measurements
for composition, but without MES). Free CO2 was kept at
200 mmol m)3 (Stumm and Morgan, 1996) using a pH controller
(a-control, Dupla Aquaristik, http://www.dupla.com) connected to a
cylinder with pressurized CO2. Shoots treated with Triton X-100
were rinsed with incubation medium prior to insertion into the
Perspex tanks. Leaves formed during the 7-day treatment period
were brushed with Triton X-100 and rinsed. Plastic mesh held
approximately 50 mm below the top of each Perspex tank prevented
emergence of any leaves. This mesh was present on all Perspex
tanks (i.e. both air controls and the submergence treatments).
Initial and final harvests were carried out to quantify leaf, sheath
and root dry masses (freeze-dried) and tissue sugar concentrations.
Plants were sampled as blocks during a 2 h window 8–10 h into the
light period. Plants were retrieved from the Perspex tanks, rinsed
with deionized water and blotted to remove surface water, then
tissues were excised and wrapped in aluminium foil, frozen in liquid
N2, freeze-dried and dry mass was recorded, before pulverisation in
a ball mill. 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 (Yemm and Willis, 1954)
and a spectrophotometer (UV-240, Shimadzu, http://www.shimadzu.
com). The reliability of the method was verified by checks on
recovery of glucose spiked into some samples immediately prior to
extraction.
Data analyses
GraphPad Prism 5.0 (GraphPad Software Inc., http://www.
graphpad.com) was used to fit a Jassby and Platt (1976) model to
the CO2 response curves. This program was also used for one- or
two-way ANOVA (with Tukey or Bonferoni post hoc tests) and Student’s t tests to compare means.
Acknowledgements
We thank the Faculty of Natural and Agricultural Sciences at the
University of Western Australia for supporting Ole Pedersen under
the Distinguished Visitors Programme, CLEAR (a Villum Kann Rasmussen Centre of Excellence at the University of Copenhagen),
Imran Malik for his assistance with the Li-Cor, Ray Scott at the
University of Western Australia Combined Workshop for his
valuable assistance, and Hank Greenway for comments on a draft
manuscript.
References
Adam, N.K. (1963) Principles of water repellency. In Waterproofing
and Water Repellency (Molliet, J.L., ed.). Amsterdam, The Netherlands: Elsevier, pp. 1–23.
Armstrong, W. (1979) Aeration in higher plants. Adv. Bot. Res. 7,
225–332.
Bailey-Serres, J. and Voesenek, L.A.C.J. (2008) Flooding stress:
acclimations and genetic diversity. Annu. Rev. Plant Biol. 59, 313–
319.
Banerjee, S. (2007) Modeling of interphases turbulent transport
processes. Ind. Eng. Chem. Res. 46, 3063–3068.
Beckett, P.M., Armstrong, W., Justin, S.H.F.W. and Armstrong, J.
(1988) On the relative importance of convective and diffusive gasflows in plant aeration. New Phytol. 110, 463–468.
Brewer, C.A. and Smith, W.K. (1997) Patterns of leaf surface wetness
for montane and subalpine plants. Plant Cell Environ. 20, 1–11.
Colmer, T.D. (2003) Long-distance transport of gases in plants: a
perspective on internal aeration and radial oxygen loss from
roots. Plant Cell Environ. 26, 17–36.
Colmer, T.D. and Pedersen, O. (2008a) Underwater photosynthesis
and respiration in leaves of submerged wetland plants: gas films
improve CO2 and O2 exchange. New Phytol. 177, 918–926.
Colmer, T.D. and Pedersen, O. (2008b) Oxygen dynamics in submerged rice (Oryza sativa). New Phytol. 178, 326–334.
Glud, R.N., Gundersen, J.K., Revsbech, N.P. and Jørgensen, B.B.
(1994) Effects on the benthic diffusive boundary layer imposed
microelectrodes. Limnol. Oceanogr. 39, 462–467.
Greenway, H., Armstrong, W. and Colmer, T.D. (2006) Conditions
leading to high PCO2 (5–40 kPa) in waterlogged-flooded soils and
possible effects on root growth and metabolism. Ann. Bot. 98, 9–
32.
Hebets, E.A. and Chapman, R.F. (2000) Surviving the flood: plastron
respiration in the non-tracheate arthropod Phrynus marginemaculatus (Amblypygi: Arachnida). J. Insect Physiol. 46, 13–19.
Jackson, M.B. (2004) The Impact of Flooding Stress on Plants and
Crops. URL Avaialble at: http://www.plantstress.com/Articles/
(waterlogging) [accessed July 2008].
Jackson, M.B. and Ram, P.C. (2003) Physiological and molecular
basis of susceptibility and tolerance of rice plants to complete
submergence. Ann. Bot. 91, 227–241.
Jassby, A.D. and Platt, T. (1976) Mathematical formulation of the
relationship between photosynthesis and light for phytoplankton.
Limnol. Oceanogr. 21, 540–547.
Jørgensen, B.B. and Revsbech, N.P. (1985) Diffusive boundary layers and the oxygen uptake by sediments and ditritus. Limnol.
Oceanogr. 30, 111–122.
Mommer, L. and Visser, E.J.W. (2005) Underwater photosynthesis
in flooded terrestrial plants: a matter of leaf plasticity. Ann. Bot.
96, 581–589.
Mommer, L., Pedersen, O. and Visser, E.J.W. (2004) Acclimation of a
terrestrial plant to submergence facilitates gas exchange under
water. Plant Cell Environ. 27, 1281–1287.
Mommer, L., Pons, T.L., Wolters-Arts, M., Venema, J.H. and Visser,
E.J.W. (2005) Submergence-induced morphological, anatomical,
and biochemical responses in a terrestrial species affect gas diffusion resistance and photosynthetic performance. Plant Physiol.
139, 497–508.
Mommer, L., Wolters-Arts, M., Andersen, C., Visser, E.J.W. and
Pedersen, O. (2007) Submergence-induced leaf acclimation:
general responses of terrestrial plant species with different
flooding tolerance. New Phytol. 176, 337–345.
Neinhuis, C. and Barthlott, W. (1997) Characterization and distribution of water-repellent, self-cleaning plant surfaces. Ann. Bot.
79, 667–677.
Pedersen, O., Vos, H. and Colmer, T.D. (2006) Oxygen dynamics
during submergence in the halophytic stem succulent Halosarcia
pergranulata. Plant Cell Environ. 29, 1388–1399.
Ponnamperuma, F.N. (1984) Effects of flooding on soils. In Flooding
and Plant Growth (Kozlowski, T.T., ed.). New York: Academic
Press, pp. 9–45.
ª 2009 The Authors
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 58, 147–156
156 Ole Pedersen et al.
Ram, P.C., Singh, A.K., Singh, B.B., Singh, V.K., Singh, H.P., Setter,
T.L., Singh, V.P. and Singh, R.K. (1999) Environmental
characterization of floodwater in eastern India: relevance to submergence tolerance of lowland rice. Expl. Agric. 35, 141–152.
Ram, P.C., Singh, B.B., Singh, A.K. et al. (2002) Submergence tolerance in rainfed lowland rice: physiological basis and prospects
for cultivar improvement through marker-aided breeding. Field
Crops Res. 76, 131–152.
Raskin, I. and Kende, H. (1983) How does deep water rice solve its
aeration problem? Plant Physiol. 72, 447–454.
Raven, J.A. (2008) Not drowning but photosynthesizing: probing
plant plastrons. New Phytol. 177, 841–845.
Revsbech, N.P. (1989) An oxygen microsensor with a guard cathode. Limnol. Oceanogr. 34, 474–478.
Sand-Jensen, K., Pedersen, O., Binzer, T. and Borum, J. (2005)
Contrasting oxygen dynamics in the freshwater isoetid Lobelia
dortmanna and the marine seagrass Zostera marina. Ann. Bot. 96,
613–623.
Setter, T.L. and Laureles, E.V. (1996) The beneficial effect of reduced
elongation growth on submergence tolerance in rice. J. Exp. Bot.
47, 1551–1559.
Setter, T.L., Kupkanchanakul, T., Kupkanchanakul, P., Bhekasut, P.,
Wiengweera, A. and Greenway, H. (1987) Concentrations of CO2
and O2 in floodwater and in intermodal lacunae of floating rice
growing at 1-2 metre water depths. Plant Cell Environ. 10, 767–
776.
Setter, T.L., Waters, I., Wallace, I., Bhekasut, P. and Greenway, H.
(1989) Submergence of rice. I. Growth and photosynthetic
response to CO2 enrichment of floodwater. Aust. J. Plant Physiol.
16, 251–263.
Smith, W.K. and McClean, T.M. (1989) Adaptive relationship
between leaf water repellency, stomatal distribution, and gas
exchange. Am. J. Bot. 76, 465–469.
Stumm, W. and Morgan, J.J. (1996) Aquatic Chemistry, 3rd edn.
New York: John Wiley & Sons.
Thomson, C.J., Armstrong, W., Waters, I. and Greenway, H. (1990)
Aerenchyma formation and associated oxygen movement in
seminal and nodal roots of wheat. Plant Cell Environ. 13, 395–
403.
Thorpe, W.H. and Crisp, D.J. (1949) Studies on plastron respiration.
IV. Plastron respiration in the coleopteran. J. Exp. Biol. 26, 219–
261.
Turney, D. and Banerjee, S. (2008) Transport phenomena at interfaces between turbulent fluids. AIChE J, 54, 344–349.
Vogel, S. (2006) Living in a physical world VIII. Gravity and life in
water. J. Biosci. 31, 309–322.
Waters, I., Armstrong, W., Thompson, C.J., Setter, T.L., Adkins, S.,
Gibbs, J. and Greenway, H. (1989) Diurnal changes in radial
oxygen loss and ethanol metabolism in roots of submerged and
non-submerged rice seedlings. New Phytol. 113, 439–451.
Woolf, D.K., Leifer, I.S., Nightingale, P.D. et al. (2007) Modelling of
bubble-mediated gas transfer: fundamental principles and a laboratory test. J. Mar. Syst. 66, 71–91.
Xu, K., Xu, X., Fukao, T., Canlas, P., Maghirang-Rodriguez, R.,
Heuer, S., Ismail, A.M., Bailey-Serres, J., Ronald, P.C. and
Mackill, D.J. (2006) Sub1A is an ethylene-response-factor-like
gene that confers submergence tolerance to rice. Nature, 442,
705–708.
Yemm, E.W. and Willis, A.J. (1954) The estimation of carbohydrates
in plant extracts by anthrone. Biochem. J. 57, 508–514.
ª 2009 The Authors
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 58, 147–156