Stomata regulate convective flow within the gas canals of the sacred lotus, Nelumbo nucifera

Abstract Lotus, Nelumbo nucifera, posses a specialised efflux organ in the centre of their leaves, which connects the gas canals in the leaves and stems with the atmosphere through the apertures of large stomata. The role of these large central plate stomata in regulating air flow through the gas canals were examined on excised leaves and on intact plants in situ. The stomata show a diurnal cycle, closing at midday then opening during the evening and night. They act as exhaust valves, releasing pressurised air from the adjacent leaf blade and gas canals when open, preventing efflux while closed. Measurements of air flow within the petiolar gas canals of the lotus leaves show different patterns in the canals connected with the central plate stomata compared with those that only connect with the leaf blade. Flow begins in the morning, flowing up the petiole to the atmosphere through the canals connected with the central plate, and down to the rhizome through those connecting only with the leaf blade. But near midday these flow patterns change abruptly, coincident with stomatal closure, and can stop or even reverse direction. This constitutes the first evidence of stomata regulating convective air flow. Possible benefits gained from this behaviour are considered.

92 Introduction The sacred lotus Nelumbo nucifera lives in flooded, anaerobic soils. Oxygenation of its submerged organs is achieved through a system of interconnected gas spaces within its petioles and rhizomes (Grosse et al. 1991). The convective ventilation of these gas spaces (called lacunae or gas canals) is achieved by humidity- induced pressurisation: a difference in water vapour pressure across a micro-porous (<0.1 μm diameter) partition in the leaf lamina causes air to diffuse into leaves against a pressure gradient (Dacey 1987). For convective ventilation to occur, pressurised air from a leaf must flow down gas canals within the petiole, through the rhizome, and vent back to the atmosphere through an efflux point. Many aquatic plants use convective flows to ventilate their submerged organs (Armstrong et al. 1992; Dacey 1980; Grosse et al. 1991), but the actual gas flow pathways are poorly understood. Tracer gas experiments have shown that the yellow water lily Nuphar luteum drives convective air flow from young pressurising leaves down to a porous rhizome and out through older non-pressurising leaves (Dacey 1981). Flows through the reed Phragmites australis and rush Eleocharis sphacelata follow a similar path to N. luteum, with pressurised air from young leaves and culms flowing through a pith cavity to a rhizome, eventually venting through old or damaged culms (Armstrong and Armstrong 1991; Brix et al. 1992; Sorrell and Boon 1994). These plants can only generate unidirectional flow, from young leaves and culms towards efflux points in damaged leaves and stems. In contrast with these simple flows, the sacred lotus has anatomical features that suggest a far more complex flow pattern, one which may also be actively regulated by the leaves. The gas flow pathway of the lotus has two features that set it apart from other pressurising plants (Matthews and Seymour 2006a). Firstly, the gas flow system is comprised of a number of discrete gas canal pairs that run throughout the bilaterally symmetrical leaves, petioles and rhizomes. The petiole contains two large pairs of canals, each pair connecting with a separate region of the leaf blade. Pairs of canals within the rhizome then connect these canal pairs with those of adjacent leaves in a specific repeating pattern (fig. 1). This arrangement allows separate gas canals within the same rhizome or petiole to channel air in opposite directions. Secondly, unlike other plants that use old, damaged leaves as vents, the lotus has a specialised vent organ - a pale-coloured plate in the centre of the leaf blade, directly above the attachment point of the petiole. Pressurised air escapes from this central plate by flowing through the apertures of large stomata scattered over its surface. The

93 apertures of these giant stomata are three times bigger than the stomata on the leaf blade (Vogel 2004). They open into an interconnected network of gas spaces which eventually merge with the larger of the two gas canal pairs within the petiole (Mevi- Schutz and Grosse 1988; Vogel 2004). The possible significance of the efflux stomata in regulating or directing flow has been briefly commented on by past researchers studying the pressurisation of lotus leaves. Dacey (1987) was the first to speculate that the stomata might act as a valve, giving the plant the ability to modify the pressure produced by individual leaves and alter flow direction. Dacey also noted that sealing the plate with silicone grease dramatically enhanced the leaf’s ability to generate a static pressure. Mevi- Schutz and Grosse (1988) examined the connections between the central plate and the gas canals of the leaf and petiole. They concluded that the gas canal pairs within the petiole must allow for two-way flow in a single leaf, since only one of the two canal pairs connects with the central plate stomata, the other connecting just with the leaf blade. They made no comment on the possible regulatory action of the stomata on flow rate or direction. As all previous observations were made on excised leaves without considering the impact of inter-leaf gas flows, only limited conclusions could be made regarding the impact of the central plate stomata on whole-plant flow regulation. The current study was undertaken to investigate the influence of the central plate stomata on pressurisation and convective air flow through the gas canals of the sacred lotus. This was done by examining the effect of central plate stomatal apertures on gas pressure within the petiolar gas canals of excised leaves, measuring the apertures of central plate stomata in the field from dawn to dusk, and relating these observations to convective flow rate and direction in situ. To appreciate the complexity of the system and the theoretical basis for gas fluxes through it, it is necessary to provide sufficient background.

Anatomical summary The lotus grows through flooded sediments as an extensive, branching horizontal stem. Individual leaves sprout at well-spaced intervals along the rhizome, arising from nodes - swellings of the rhizome that produce a leaf, adventitious roots and an axillar shoot. Interconnecting gas canals run through all parts of the lotus except the adventitious roots and flowers. The gas canal system comprises multiple pairs of gas canals, with a complete independent set of canals running on each side of the plane of symmetry that divides the long axis of rhizome growth (fig 1c). This

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Fig. 1. Gas canal anatomy of Nelumbo nucifera. Cross-sections of the petiole (A) and rhizome (B) showing gas canal pairs. (C) Cutaway diagram showing the three chambers in the node connecting the gas canals of the rhizome with those of the petiole, as well as the connection between the A canal of the petiole and the porous central plate of the leaf. (D) Schematic of gas canal connections between three adjacent leaves. The two grey lines indicate potential flow directions down two pressurising leaves and up one vent leaf (Matthews and Seymour 2006a).

95 separation continues at the leaf with each half of the divided central plate connecting with one half of the gas canal pairs. The number of canal pairs differs between the petioles and rhizomes. The petiole has two major canal pairs (A and B) with two minor pairs (c and d) (fig. 1a). The A, c and d canals from each half of the rhizome interconnect beneath the central plate, also joining with approximately two-thirds of the leaf blade’s gas canals (fig. 1c). The B canals do not communicate with the central plate, but merge with the remaining leaf canals (Mevi-Schutz and Grosse 1988; Vogel 2004). The rhizome has three major pairs of canals (I, II and III), with two individual canals (IV and V) lying on the plane of symmetry (fig. 1b). The gas canals of the petiole and rhizome are united by three pairs of large cavities (X, Y and Z) within the node. Caverns Y and Z are a junction between the canals of the petiole and rhizome, while cavern X interconnects only the canals of the rhizome. The caverns unite the gas canals in such a way that pressurising air from a B canal must flow through at least one adjacent node before it can reach an A canal that vents back to the atmosphere through the stomata of a central plate (fig. 1d).

Relationship between leaf pressure and efflux resistance An excised lotus leaf without an efflux point (i.e., with a blocked petiole and sealed central plate) generates a static pressure differential (ΔP) proportional to the difference in water-vapour pressure between the inside (Pwi) and outside (Pwa) of its leaf:

ΔP = − PP wawi (1) (Dacey 1987). Therefore, the pressure produced by a leaf with closed central plate stomata approaches ΔP. If the central plate stomata open, the pressurised air within the leaf flows back to the atmosphere, and this causes the pressure differential to drop.

Therefore, by keeping Pwa constant, it is possible to relate decreases in ΔP to decreases in central plate resistance. The relationships between leaf pressure, central plate stomatal resistance, and flow, can be established using an electrical analogy (fig. 2). The leaf blade converts a difference in molar fractions of gases across its micro-porous partition into a force (ε) equal to Pwi - Pwa. This is similar to the behaviour of a galvanic cell, where a difference in charge between two separated metals produces an electromotive force. By keeping the leaf in a constant temperature and humidity environment, ε remains constant. Like all batteries, the leaf also has an internal resistance (r), which is due to

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Fig. 2. Circuit analogy describing pressurisation and flow through a single lotus leaf with blocked petiolar gas canals. A difference in water-vapour pressure drives a diffusive influx of air (ε) through the resistance of a micro-porous partition (r). This drives air flow (I) through the variable resistance of the central plate stomata (R), causing a pressure gradient (P). the resistance of its micro-pores to diffusion. The pressure potential developed by the leaf blade drives a current of air (I) through the variable resistance of the central plate stomata (R), resulting in a pressure difference (P). Because of the pores’ resistance, the flow rate can never exceed a maximum rate (Io) given by ε I = (2) o r In this situation there is no efflux resistance, and the rate of flow generated by ε is limited only by the micro-porous partition’s resistance to diffusion r. However, the actual flow rate will always be lower than this, as R is in series with r: ε I = (3) + Rr Ohm’s law describes the relationship between P, I and R, with the pressure drop across a resistance equal to P = IR (4) Therefore, changes in R due to the action of the central plate stomata alter the measured pressure difference P according to ⎛ ε ⎞ P = ⎜ ⎟R (5) ⎝ + Rr ⎠ And since both ε and r are constants, as R increases towards infinity, P increases asymptotically towards a maximum value (Pmax) determined by ε, while I behaves in the opposite manner, decreasing asymptotically to 0 (fig. 3). Knowing this relationship, it can be said that: P ∝ R (6)

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Fig. 3. Relationship between the resistance of the central plate stomata efflux point (R), flow rate (I) and the pressure differential (P). As stomatal resistance (R) increases the pressure (P) rises asymptotically towards the maximum static pressure (Pmax), while flow rate (I) falls to negligible levels.

And therefore the measured P is an indirect measure of the resistance of the central plate stomata. Using this relationship, this study examines the role of central plate stomata in regulating pressure and flow in the gas canals of the lotus by measuring changes in leaf pressure in vitro and diurnal changes in stomatal apertures in the field.

Materials and Methods Measurement of efflux regulation Changes in the efflux resistance of central plate stomata were measured in excised emergent lotus leaves (n = 11) collected from an open-air pond in the Adelaide Botanic Gardens, South Australia. On the morning they were to be used (before 09:00), mature fully-expanded leaves were covered with large polyethylene bags before their stems were cut below the waterline. Their cut ends were kept submerged and the leaves were transported quickly to the laboratory (~ 15 min), and kept in darkness until required. Leaves treated in this way showed no signs of wilting. Before a leaf was used, its petiole was trimmed to 35 cm while underwater and then secured in a custom-made leaf chamber 33 × 33 × 8 cm (fig. 4). The petiole was threaded through a 1 cm diameter hole in the floor of the chamber and into a measuring cylinder filled with water. The gap around the petiole was then sealed with putty (Blu-tak, Bostik, UK). Two calibrated T-type 38 gauge thermocouples were used to measure the temperature of the top and bottom surfaces of the leaf approximately 10 cm from the centre. The thermocouples were connected to a

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Fig. 4. Apparatus for observing changes in ΔP, measured within the petiole’s A canal. Air is drawn from the room by a pump (A) through a mass flow regulator (B) and then bubbled through a bottle of water (C) to saturate it with water vapour. The saturated air is then passed through a Peltier condenser (D) regulated by a humidity controller (E) to produce a constant RH air stream. This air is passed over the upper surface of a lotus leaf secured in the leaf chamber (F). The leaf’s petiole is kept submerged in a measuring cylinder of water (G) which keeps the leaf hydrated and blocks the petiole’s gas canals, allowing the static leaf pressure to be measured by a pressure transducer (H). Light is applied to the central plate from a lamp suspended above the chamber (I). A RH sensor (J) measures the humidity of the air leaving the chamber. The temperature of the upper and lower leaf surfaces are measured using T- type thermocouples connected to a thermocouple preamplifier (K), and the outputs of the preamplifier, RH sensor and pressure transducer are all measured with an analogue to digital converter (L) and recorded on a desktop computer (M).

99 thermocouple preamplifier (Sable Systems, Nevada, USA). The chamber was then sealed with a plastic lid with a large (33 × 33 cm) polyethylene window. The pressure differential generated by the leaf was measured within the A gas canal by threading 30 cm of a 65 cm length of 0.8 mm internal diameter PVC tubing up the gas canal through the cut end of the petiole. In this way the end of the tube was placed inside the gas canal 5 cm below the central plate and well above the water in the measuring cylinder below. The free end of this tube was then connected to a PT5 volumetric low pressure transducer (Grass Telefactor, West Warwick, USA) using a 0.8 mm (21 G) needle. The reference side of the transducer was connected to the leaf chamber by a short length of 50 mm tubing, allowing the pressure transducer to measure the pressure differential between the leaf blade and the chamber. Air was then blown through the chamber at 7 L min-1 from a pump (Reciprotor, Copenhagen, Denmark) connected to a mass flow controller (Aalborg, New York, USA). The relative humidity (RH) of air being blown into the chamber was regulated using a DG-3 RH controller (Sable Systems, Nevada, USA). The RH of the air exiting the chamber was measured with a sensor (50Y Humitter, Vaisala, Vantaa, Finland). A spotlight illuminating an area of approximately 12 cm2 was projected onto the central plate from an adjustable halogen lamp (2050-HB, Heinz Walz GmbH, Germany), the photon flux density (PFD) of which was monitored using a quantum sensor (LI-190, LiCOR, Nebraska, USA) attached to a data logger (LI-1400, LiCOR, Nebraska, USA). All this equipment, including the leaf chamber, was maintained at 25 ± 1 °C within a constant temperature cabinet. The voltage outputs from the thermocouple preamplifier, pressure transducer and humidity meter were all recorded on a desktop computer at 1 Hz (4/30 Powerlab and Chart software, ADInstruments, Sydney, Australia). Leaves placed within the chamber were given a 20 min settling time at 60 % relative humidity. RH was then decreased to 30 % for the duration of the experiment. This value is slightly higher than the RH measured in the field, which routinely dropped to ~20 % at midday. The lamp was turned on 20 min after this RH decrease, and a PFD of 300 μmol m-2 s-1 was applied to the central plate. The PFD was increased by 200 μmol m-2 s-1 every 20 mins in a stepwise fashion until a maximum of 2100 μmol m-2 s-1 was reached, equal the average maximum PFD measured in the lotus pond. The PFD was then reduced three times in 200 μmol m-2 s-1 increments at 40 min intervals and then twice in 400 μmol m-2 s-1 increments until a PFD of 700

100 was reached, after which the light was switched off. After a further 20 mins the central plate was sealed with petroleum jelly. Control runs were also performed with the leaves kept in darkness and exposed to the same humidity regime as treatment leaves.

Casting of central plate stomata Central plate stomata on emergent lotus leaves growing in an open air pond at the Adelaide Botanic Gardens were examined for diurnal changes in aperture. Sampling was carried out over three non-consecutive days between January and February 2007. The first casts were made pre-dawn between 06:00 and 06:30, and casts were then taken at 2-h intervals until 20:00 for a total of 8 sample periods per day. Casts were taken from 10 randomly selected, mature leaves during each sample period. Ambient air temperature and PFD within the pond were also measured at each sample period using a hand-held Fluke model 52 digital thermometer (Washington, USA) and a horizontally mounted quantum sensor (LI-190, LiCOR, Nebraska, USA) attached to a data logger (LI-1400, LiCOR, Nebraska, USA). The central plate stomata were cast by applying freshly mixed polyvinylsiloxane dental impression material (President light body impression material, Coltène Whaledent, Switzerland) to the central plate with a spatula. After approximately 5 to 10 min, depending on the ambient temperature, the set cast was peeled from the leaf. Leaf temperatures were then measured using the digital thermometer, with the tip of the 38 gauge thermocouple resting on the upper surface of the leaf blade approximately 10 cm from the central plate. A second series of casts were made in December 2008 to determine if central plate stomatal apertures changed with age. Newly emergent, furled and partially unfurled leaves were marked with flagging tape at weekly intervals over 4 weeks. Each age group consisted of 12 leaves. The central plates were cast before dawn (between 05:00 and 06:00) on the morning of the 5th week. A thin coating of clear nail polish was applied to one half of each of the negative polyvinylsiloxane casts to produce a positive cast. The positive cast was mounted beneath a cover slip on a microscope slide and examined with a light microscope (Anax, Olympus, Japan) at 100× and 400× magnification. Photos of the casts were taken using a digital camera (Canon EOS 350D digital SLR) connected to the eyepiece of the microscope by an adaptor (MaxView™, ScopeTronix, www.scopetronics.com). The digital photos were analysed using Scion Image

101 software (Scion Corp., Maryland, USA), with the stomatal dimensions of aperture length (parallel to the long axis of the guard cells) and width (perpendicular to the long axis of the guard cells, at the widest point) taken from five stomata in each cast.

Measurement of flow direction within the petiolar gas canals Measurements of gas flow rate and direction were made on lotus leaves growing in the Adelaide Botanic Gardens. Either an A or a B petiolar gas canal was opened by making a steep, upward insertion into the petiole with the tip of a 1.65 mm diameter (16 ga) needle. The needle was pulled away from the petiole at a right angle, opening the gas canal with a ~6 mm long slit. This slit allowed two 18 cm lengths of 1.52 mm external and 0.82 mm internal diameter polyethylene tubing with male Luer-loc ends to be inserted into the gas canal, one threaded up the stem and the other down. The tubes were securely taped to the petiole and the slit was then backfilled with petroleum jelly (Vaseline, Lever Rexona, Australia) from a syringe, creating an air-tight seal around the tubing and completely blocking the gas canal. Mass flow meters (0-10 mL min-1, Aalborg, New York, USA) mounted atop 1.8 m wooden stakes were positioned next to the leaves. They were connected to the implanted tubes by 40 cm lengths of 4 mm ID PVC tubing with female Luer-loc connectors. As it is only possible for flow to be measured in one direction, the connections between the petiole and flowmeter were reversed after several days to record flow both up and down the gas canal. The flow meters were powered from a single 12 V deep-cycle battery, and flow rate was recorded at 1 min intervals with a Squirrel data logger (Grant Instruments, Cambridge, U.K.). After the measurements were made the petiole was collected with the implanted tubes in place, and both the position of the tubes within the gas canal and the soundness of the petroleum jelly seal were examined.

Measurement of photosynthetic yield A simple experiment was performed to determine if a difference in photosynthetic activity exists between areas of the leaf lamina connected with either A or B canals. Two groups of eight mature, emergent lotus leaves were marked with flagging tape. Petroleum jelly was injected into the petioles of one group of leaves to block their A canals, while the other group was left as a control. The two small holes made in the petiole did not appear to affect the treatment leaves, as there was no sign of wilting and the leaves remained healthy for weeks following this manipulation.

102 Photosynthetic yield was then measured from areas of the leaf lamina which connect with either the A or B canals using a photosynthesis yield analyser (MINI-PAM, Heinz Walz GmbH, Germany). Four measurements, two on the A lamina and two on the B lamina, were made on each leaf at two hour intervals from 08:00 until 20:00. Some treatment leaves were cut following measurement to verify that blockage of the A canals had been achieved.

Results Data are given as the means of sample size n ± 95 % confidence intervals or standard deviation.

Effect of central plate stomata on gas canal pressures The action of the central plate stomata has an obvious effect on the pressure measured within the A canals of the petiole. Gently blowing air across the leaf blade always caused bubbles to emerge from the cut end of a submerged petiole through the B canals, but only occasionally from the A canals. Nitrocellulose casts of the central plate stomata from leaves with bubbling and non-bubbling A canals revealed that the central plate stomata of bubbling A canals were always closed. Artificially closing the stomata by sealing the central plate with petroleum jelly produced the same pattern of efflux from the cut end of its petiole as when the stomata were closed, i.e., both the A and B canals bubbled. Scoring the surface of the central plate with a razor blade stopped the A canals from bubbling but did not affect B canals.

Central plate stomatal apertures The mean diameter of the central plate was 14.3 ± 0.7 mm (n = 140), but it varied considerably depending on leaf size, the largest plate measured being over 22 mm in diameter, the smallest closer to 7 mm. Regardless of its size, casts of the plate revealed that stomatal density was consistent between leaves, occurring at a frequency of 63 ± 2 mm-2 (n = 80). The stomata had mean aperture lengths of 27.3 ± 0.3 μm (n = 1190). Fully open stomata were found to have aperture widths of 30 μm or greater, making their apertures appear circular (fig. 5). Assuming the stomata were maintaining circular pores, their combined area would account for approximately 3.7 % of the total surface area of the central plate. The largest central plates would therefore have a total vent area of approximately 14 mm2.

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Fig. 5. Nitrocellulose casts of the petiolar plate, showing A. stomatal density, B. closed stomata and C. open stomata.

A diurnal pattern of stomatal opening was clearly apparent from the casts. Many central plates had open stomata early in the morning, but all stomata were fully closed by midday, opening again over the course of the afternoon and evening (fig. 6). This pattern recurred over three clear, sunny days, with stomatal apertures at midday uniformly closed (3.5 μm ± 1.4), as indicated by the very low standard deviation recorded at this time. The large standard deviations either side of midday indicated a considerable degree of variation within this basic pattern, with the central plate stomata of some leaves closed at every sampling period. Leaf age was shown to play no significant role in determining pre-dawn stomatal apertures, with both young and old leaves likely to possess either open or closed stomata (fig. 7).

Fig. 6. Diurnal changes in the aperture width of central plate stomata and concurrent PFD. Circles indicate the grand mean at each time period. Error bars at each point indicate ± SD of 150 stomata from 30 leaves.

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Fig. 7. Pre-dawn (05:00 – 06:00) stomatal aperture widths measured from the central plates of leaves ranging in age from 1 to 4 weeks old. There are no significant differences between age classes. Error bars at each point indicate ± SD of n = 12, 10, 12, 9 leaves from the 1, 2, 3 and 4 week-old age groups, respectively.

Effect of light on central plate stomata and gas canal pressure The response of the central plate stomata to increasing PFDs varied between leaves (n = 11), with three main patterns observed. Leaves which began the experiment with open central plate stomata (n = 3) produced saw-shaped pressure curves over the course of the experiment (fig. 8a). The pressure within the A canal began to rise once the chamber humidity had dropped to a stable level, increasing rapidly when the central plate was illuminated. The rate of pressure increase slowed towards the maximum PFD of 2100 μmol m-2 s-1. The first 200 μmol m-2 s-1 decrease in PFD initiated a steady decline in pressure which declined to its lowest level once the light was turned off. Subsequent application of petroleum jelly to the central plate then caused the measured pressure to rise immediately to a level near the maximum pressure reached during the experiment. Several other leaves which began the experiment with closed or partially closed stomata as determined by observing the emergence of bubbles from their petioles (n = 3), also showed an initial increase in pressure when exposed to increasing PFDs. However, once these leaves were exposed to PFDs above 1500 μmol m-2 s-1, the pressures within their A gas canals began to oscillate (fig. 8b). The amplitude of the pressure oscillations began to

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Fig. 8. Static leaf pressure (ΔP) from two leaves in a constant temperature/relative humidity environment. PFD was varied in a stepwise fashion during the experiment. ΔPmax indicates estimated maximum static pressure. Asterisks (*) indicate when the central plates were sealed with petroleum jelly. Leaf A began the experiment with open central plate stomata, B began with closed stomata. decrease once the PFD was reduced, as seen by the declining minimum and maximum pressures of each oscillation. Despite the declining pressure, the oscillations occurred at regular intervals, with a mean periodicity of 20 min. A single leaf which began the experiment with open stomata began to produce pressure oscillations shortly after exposure to a PFD of 900 μmol m-2 s-1, and continued for the duration of the experiment. The amplitude of the pressure oscillations tended to be erratic, initially increasing, and then declining at the higher light levels, before again increasing as the light was reduced. Turning off the light caused a small, transient rise in pressure before it declined to negligible levels. The oscillation frequency of this leaf was only half that of the other leaves, cycling at an average of 40 min peak to peak. Some leaves showed no response at all to the increases in light (n = 5), maintaining a pressure barely above 0 and showing an insignificant pressure increase when the central plate was sealed. All these leaves showed signs of wilting, with their edges turning a pale green and beginning to curl. Control leaves (n = 4) kept in darkness but exposed to the same humidity as illuminated leaves maintained a low constant pressure which did not change during the experiment. The observed changes in pressure were not affected by heat from the halogen lamp, as it only illuminated the central plate and caused a negligible (< 0.2 °C) increase in leaf blade temperature. Small, regular pressure fluctuations were caused by the on/off cycling of the thermostat in the constant temperature cabinet. However,

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Fig. 9. Rates of flow up an A canal (solid line) and down a B canal (dashed line) measured in two separate leaves over two consecutive days. The decreases (indicated by arrows) are natural occurrences. these small pressure variations were readily distinguishable from the larger pressure fluctuations caused by the central plate stomata.

Flow rate and direction Convective flow in both the A and B gas canals began in the morning when air temperature began to rise (fig. 9). The flow rate then increased sharply once sunlight began to warm the leaves. Maximum flow rate in the A canals was observed between 14:00 – 16:00. Flow then decreased at sunset, but persisted at very low levels until around 00:00, at which time it became undetectable until the following morning. While flow up A canals generally followed a ‘bell-curve’ pattern over the course of a day, flow down the B canals appeared to be bi-modal, increasing in the morning, decreasing to very low levels around midday before again increasing towards evening. However, there was some variation in these flow patterns. Air flow up the A canals was occasionally found to drop sharply between 11:30 and 12:00, temporarily decreasing to zero in measurements on three separate leaves. After < 15 to 30 min, air once again began to flow up the A canal. The drop in air flow measured in the B canals also occurred at midday, but instead of dropping rapidly, the flow rate declined slowly and stayed low for up to 2.5 h. Reversing the connection between the gas canal and flow meter showed that the direction of flow in the B canal could reverse during the afternoon, with flow proceeding up the canal (fig. 10). Flow reversals in

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Fig. 10. Rate of flow down the B canal (solid line) and up the same B canal (dashed line) measured the following day.

Fig. 11. Flow measured up an A canal (solid line) and relative humidity (dashed grey line) over two consecutive days. On the first day the leaf shows a substantial natural drop in flow during the afternoon. At 2:05 on the second day the central plate was sealed with petroleum jelly (asterisk), causing an immediate drop in the rate of air flow up the A canal.

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Fig. 12. Photosynthetic yield measured from treatment leaves (white symbols) with blocked A canals and control leaves (filled symbols). Measurements were made on areas of the leaf lamina connecting with the A canals (circles) and B canals (squares) as indicated on the lotus leaf (inset). Differences either between controls and treatments or between A and B lamina areas on control leaves were not significant. Each point is the mean of 16 measurements from 8 leaves. Error bars are 95 % CI. the A canal were far less obvious, with only very brief spikes in flow rate (less than 2 mL min-1) down the A canals detected after 12:00. Flow interruption episodes in both A and B canals did not occur every day. The effect of sealing the central plate of a single leaf on flow up an A canal was recorded. Application of petroleum jelly instantly reduced the rate of flow up the A canal by half, but did not stop flow altogether (fig. 11).

Photosynthetic yield The data show that between 12:00 and 16:00 the mean yield of treatment leaves dropped below that of the control leaves, but a t-test showed that the difference was not statistically significant (fig. 12). A paired t-test also revealed no significant difference between the yields measured from the A and B sections of the control leaf lamina at any time.

Discussion These experiments demonstrate the unique function of the central plate stomata of N. nucifera. Whereas the stomata of all other leaves function to regulate

109 the inward diffusion of carbon dioxide while minimising the outward diffusion of water vapour, the stomata of the lotus central plate have been modified to act as exhaust valves capable of regulating the efflux of pressurised air from gas canals. The observations and experiments made on the excised leaves show that when the central plate stomata are open, they are the path of least resistance for the efflux of pressurised air. Because the A canals connect with both the stomatal apertures and a large portion of the pressurising leaf blade, pressurised air from the leaf blade can escape when the stomata are open. Thus the pressures measured within the A canals are low. But when the stomata close, or are artificially sealed with petroleum jelly, the pressure within the A canals rises dramatically (fig. 8). The regulatory action of the stomata is shown by the changes in pressure measured when the central plate was illuminated by varying PFDs. As the leaf blade’s pressurising capacity is held constant in a stable temperature and humidity environment, a leaf with a fixed resistance efflux point would pressurise to a constant level. This was not the case, with leaves showing rapid increases and decreases in pressure when exposed to stepped increases and decreases in PFD. Some leaves also displayed pronounced pressure oscillations which are attributable to synchronous oscillations of the stomata, which could be caused by the rapid increase light levels or low xylem conductance (Prytz et al. 2003). While it was not the purpose of this study to specifically examine stomatal oscillation, it does reveal that the apertures of the central plate are actively regulated, cycle synchronously, and have a strong influence on efflux from the leaf. The response of the central plate stomata to varying levels of light correlates well with the measurements of central plate stomatal apertures made from lotus leaves growing in an open-air pond. The stomatal casts show that the apertures were larger on average during periods of low light, and all were closed at midday shortly before the daily maximum PFD was reached. Once light level began to decline, there was a coincident rise in average aperture width. Interestingly, the stomata on the leaf blade do not follow this pattern, but maintain open apertures throughout the day (Takagi et al. 2006). Unlike the yellow waterlily, which vents through its older leaves (Dacey 1981), the central plate stomatal apertures measured from older lotus leaves were no more likely than young leaves to have their stomata open and act as efflux points. The daily closure of central plate stomata had an effect on the pattern of flow recorded within the petioles of the lotus leaves. On several occasions, flow up the A canals abruptly stopped at midday, while flow down the B canals declined or reversed

110 (Fig. 9). The timing of these events corresponds with the midday decrease in stomatal aperture, and constitutes the first in situ evidence of stomatal regulation of convective flow. Assuming no change in efflux resistance, it would be expected that flow rates would reach their peak around this time, as low ambient water vapour pressure and high leaf temperatures would produce the highest pressures to drive the fastest convective flows. The different rates of flow change in the A and B canals appear to be related to the gas canal anatomy of the lotus. A leaf with open central plate stomata acts as an efflux point, allowing pressurised air from adjacent leaves to flow from their interconnecting rhizome, up its A gas canals and vent to the atmosphere. Therefore the rate of flow up an A canal is determined by the resistance of the leaf’s central plate stomata, with rapid closure of the stomata able to cause an equally rapid decrease in flow. Flow down the B canal, however, is determined by more than one central plate. Pressurised air flowing down a B canal enters canal III of the rhizome, which then connects with the A canals of every leaf growing from the same rhizome. Thus the rate of flow down the B canal is regulated by the parallel resistances of multiple central plate efflux points. As the stomata on the separate central plates are likely to be closing at different rates, so the total efflux resistance decreases slowly, and increases just as slowly (Fig. 9). Although the central plate stomata play an active role in the convective ventilation of the lotus, their adaptive benefit is not entirely clear. If ventilation of the rhizome is taken to be the only goal of leaf pressurisation, then any down-regulation of the flow could be considered to be a disadvantage. From this perspective there are several puzzling aspects when considering the role of the central plate stomata. First, because the central plate stomata connect with both the A canals of the petiole and the majority of the leaf blade’s gas canals, when they open to allow airflow up from the rhizome, they simultaneously vent the pressurised air generated by two thirds of the adjacent leaf blade directly to the atmosphere. Thus the central plate effectively short circuits the majority of the pressurising leaf surface that could otherwise contribute to flow. The second problem is that when the stomata close, they could stop convective flow entirely, as all petiolar gas canals would then be connecting with pressurising leaf blades. There is, however, a potential benefit to be gained by this behaviour. It has been demonstrated that several wetland plants take advantage of the large amounts of

CO2 found in flooded sediments by transporting it up through their gas canals and into their leaves to be used in photosynthesis (Brix 1990; Constable and Longstreth 1994;

111 Dacey and Klug 1982a; Singer et al. 1994). Given that CO2 is potentially limiting to photosynthesis (Longstreth 1989), the lotus would appear to be well situated to take advantage of this resource, otherwise having access to unlimited water and growing in full sunlight.

For lotus to use the sediment-derived and respiratory CO2 for photosynthesis, it must be moved from the rhizome into the leaf lamina. If the air within the buried gas canals and rhizomes were vented up a petiole and out into the leaf blade instead of out through the central plate then it would be readily available for photosynthesis. This could happen when the stomata close at midday. As multiple leaves grow from a single rhizome, so the direction of flow between them is the result their individual pressures and efflux resistances. As seen from the stomatal casts (fig. 6), the only times when any of the central plate stomata are completely open is during the morning and late afternoon. Therefore, those leaves with open central plates act as vent points when pressurisation begins during the morning, offering the path of least resistance for the efflux of air. The closure of the central plate stomata at midday would not only prevent this efflux, but connect the largest pressurising leaf surface to the A canals. With all gas canals leading to leaf blades, the direction of flow is from leaves generating higher pressures to those generating lower pressures, the air flowing into the leaf and venting out through its upper surface. As the pressurised air must have first passed through the convoluted gas canals in the rhizome, it would be enriched with CO2 which would then be delivered directly into the leaf blade and to the site of photosynthesis. This must have been occurring when flow was recorded proceeding up the B canal (fig. 9), as this canal only connects with the leaf blade, and not the central plate. Similarly, gas flowing up the A canal must have vented through the leaf blade when the central plate was sealed with petroleum jelly, as preventing efflux only halved the rate of flow without stopping it entirely (fig. 11). While experiments to determine if air from the gas canals increased photosynthesis were not conclusive, the yield reduction measured from leaves with blocked gas canals is intriguing (fig. 12). Examination of the gas composition within the lotus’ canals using more sensitive techniques could yield more robust data about possible sediment to leaf CO2 fluxes and gas flow directions. The behaviour of the central plate stomata could also be beneficial in other ways. By opening in the morning, fresh air would be flushed through the oxygen- deprived rhizome as soon as the leaves began to pressurise. By midday, when the PFD is highest and capacity of the leaves for pressurising and photosynthesis are both

112 at their peak, the stomata close, creating the high pressures necessary for forcing CO2- enriched air from the rhizome through the leaf lamina. Then towards evening the stomata open again, ensuring that any small pressures generated during the night is converted into a convective flow to aerate the rhizome.

Acknowledgments We sincerely thank the Adelaide Botanical Gardens for supplying plant material for the laboratory work and providing access to Nelumbo Pond.

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