Spatio-temporal changes of photosynthesis in carnivorous plants in response to prey capture, retention and digestion

Abstract

Carnivorous plants have evolved modified leaves into the traps that assist in nutrient uptake from captured prey. It is known that the traps of carnivorous plants usually have lower photosynthetic rates than assimilation leaves as a result of adaptation to carnivory. However, a few recent studies have indicated that photosynthesis and respiration undergo spatio-temporal changes during prey capture and retention, especially in the genera with active trapping mechanisms. This study describes the spatio-temporal changes of effective quantum yield of photochemical energy conversion in photosystem II (Φ_PSII) in response to ant-derived formic acid during its capture and digestion.

Carnivorous plants have evolved their leaves into the modified structures called traps, which assist in nutrient uptake from prey bodies.¹ The traps attract, catch and digest the animal prey; however, some species obtain substantial amount of nutrients from leaf litter (Nepenthes ampullaria), algae (Utricularia) or from faeces of tree shrew Tupaia montana (Nepenthes lowii, N. rajah, N. macrophylla) as a result of adaptive radiation with regard to nitrogen sequestration.^2–5 Carnivorous plants are mainly restricted to sunny, moist and nutrient-poor environment, because only in this environment would the cost of producing traps be lower than the benefits gained from prey.⁶ From carbon metabolism point of view, the benefit is in term of increased rate of photosynthesis per unit leaf mass as a result of increased nitrogen concentration in the leaf or an increase in the total leaf mass that can be supported.^6–8 The costs of carnivory include reduced rate of net photosynthesis (A_N) in traps as a result of leaf adaptation to carnivory or increased rate of respiration (R_D) as a result of extra energy requirements for attracting, capturing and digesting the prey.⁹ Whereas the reduced A_N in the traps has been confirmed several times, the higher R_D in traps is still ambiguous.^9–12

Until now studies assessing the cost of carnivory have usually been confined to measurements of A_N and R_D in carnivorous traps vs. non-carnivorous leaves, to the construction costs and payback times for carnivorous organs or to the carbon costs of sticky mucilage secretion by glands.^9–16 There is a growing body of evidences that prey-catching is active process involving spatio-temporal changes in A_N and R_D in traps, at least in carnivorous plants with active trapping mechanisms.¹⁷ First evidence, however not convincing, came from the work of Knight.⁹ She found that bladders of aquatic bladderwort Utricularia macrorhiza had a slightly greater R_D (10%) than assimilation leaves, but these differences were not significant. Later Adamec found that R_D of bladders in six Utricularia species was 75–200% greater than that in the leaves.¹⁸ The action of Utricularia bladder is one of the fastest movement in plant kingdom. When the trap of Utricularia is set, ready for trapping, it looks shrunken due to negative hydrostatic pressure. When the trapdoor is stimulated by prey it opens, sucks the water with prey and the door rapidly shuts. This firing process takes about 30 ms. Then the bladder restores its negative hydrostatic pressure by the removal of water from trap lumen through the glands. The resetting of bladders is a respiration-dependent process accompanied by the consumption of ATP.^1,19,20 Adamec suggests that the bladders of Utricularia were in post-firing state and were therefore pumping water and is possible that their R_D in this state was much higher than in their resting state.¹⁸ Adaptative changes in cytochrome c oxidase in the genus Utricularia may provide respiratory power for bladder function.²¹ The most famous carnivorous plant the Venus flytrap (Dionaea muscipula) also uses active trapping mechanism for prey capture. Recently, Hájek and Adamec published that the traps of D. muscipula had lower A_N, whereas the R_D in lamina and trap was comparable.¹² This is in accordance with the classical interpretation of cost/benefit model of carnivory. However, in our previous study we have shown that trigger hair irritation in the open as well as in closed trap of Dionaea muscipula resulted in the rapid increase of R_D and decrease of effective quantum yield of photochemical energy conversion in photosystem II (Φ_PSII).¹⁷ We have suggested that this is a result of generation of action potentials upon trigger hair irritation.^22–25

The link between electrical signals and inhibition of photosynthesis and stimulation of respiration has been described in several plant species, however it has not been known in carnivorous plants.^26–30 Another genus of carnivorous plants that generates action potentials in response to mechanical irritation is sundew (Drosera). In the Dionaea traps, the action potential originates in any one of the six trigger hair and the potential propagates over the entire trap blade more rapidly across the lower (abaxial) surface. In the Drosera tentacle, action potentials are initiated by a receptor potential just below the swollen head of the tentacle and propagate only to its base and do not reach the leaf lamina.^31–33 This is in accordance with the results that separated D. prolifera tentacles have many times greater R_D in comparison with that of leaf lamina. This proves a very high metabolic and physiological activity of tentacles probably as a results of electrical irritability.¹¹ It has been suggested that at least some of the energy connected with the rise of R_D after action potential is utilized for the restoration of the state of ionic balance (i.e., restore the resting state).²⁶ Except the electrical signals, chemical substances seem to be also effective in effecting photosynthesis in carnivorous plants. This study describes negative impact of ant-derived formic acid on effective quantum yield of photochemical energy conversion in PSII (Φ_PSII), which is a sensitive indicator of plant photosynthetic performance.

We measured the chlorophyll fluorescence in response to prey capture (ant Lasius niger L.) by the leaf of Drosera capensis L. Ten one-year-old Drosera capensis L plants were grown in growth chamber at a irradiance 200 µmol m⁻² s⁻¹ photosynthetic active radiation (PAR), 14/10 h day/night cycle and daily temperature ∼25°C. Before the measurement, the plant was adapted to light intensity 100 µmol m⁻² s⁻¹ PAR for 10 minutes (time required for steady state values of chlorophyll fluorescence in light-adapted state, F_t). The actinic light was provided by fluorcam FC-1000 LC (Photon System Instruments, Czech Republic) using red emitting LED diodes (λ = 620 nm). The experiment started by application of first saturation pulse (4,000 µmol m⁻² s⁻¹ PAR, 800 ms duration, λ = 620 nm). Then (after 10 seconds) one ant (Lasius niger) was gently put on the D. capensis leaf. During the first two hours, saturation pulses were applied every three minute, thereafter every hour and later every 24 hour. After each saturation flash the visible pictures were taken by camera Nikon D60 (Nikon, Thailand). The Φ_PSII, which indicates the proportion of the light absorbed by chlorophyll associated with photosystem II that is used in photochemistry, was calculated as (F_m′ - F_t)/F_m′.^34,35 The experiment was repeated without ant's abdomen (the abdomen was cut by scalpel but ants survive and their moving was not affected). In the last experiment 1 µL 15 M formic acid (Fluka) was dropped on the leaf. All experiments were repeated four times independently and data presented are representative.

The inhibition of Φ_PSII occurred within a few minutes after the ant was trapped by Drosera tentacles and then again after 96 hours (Fig. 1). Repeating the experiment without ant's abdomen had no negative impact on Φ_PSII in spite of ant-induced leaf folding (Fig. 2). The most common substance in ant's abdomen is formic acid.³⁶ This indicates that the inhibition of Φ_PSII was caused first by the spraying of the formic acid on the leaf by the struggling ant and then by releasing the formic acid after 96 hours from ant's abdomen as a result of digestion. Therefore, the preliminary observation in Drosera mentioned at the end of discussion of our previous study was not associated with electrical signals.¹⁷ The inhibition was caused by the ant Lasius niger, which inhibits the Φ_PSII in D. capensis by releasing the formic acid from its abdomen. This is consistent with the findings that propagation of action potentials in Drosera is restricted only to the tentacles and therefore had no effect on photosynthesis in leaf blade (Fig. 2). Further, application of 1 µL 15 M formic acid resulted in very similar effect like the living ant with intact abdomen (Fig. 3). The concentration of formic acid was chosen according to data that the venom of Formica rufa contains 5–17 M formic acid.³⁶

Figure 1.

The visible response (A) and the response of effective quantum yield of photochemical energy conversion in photosystem II (Φ_PSII, B) of Drosera capensis leaf to prey capture (intact ant Lasius niger). The ant was put on the leaf in time 10 seconds.

Figure 2.

The response of effective quantum yield of photochemical energy conversion in photosystem II (Φ_PSII) of Drosera capensis leaf to prey capture (ant Lasius niger without abdomen). The ant without abdomen was put on the leaf in time 10 seconds. Note that no changes in Φ_PSII occurred in spite of leaf folding.

Figure 3.

The response of effective quantum yield of photochemical energy conversion in photosystem II (Φ_PSII) of Drosera capensis leaf to 1 µL of 15 M formic acid. The drop of formic acid was put on the leaf in time 10 seconds.

The production of formic acid by ants is thought to have evolved to improve capture of invertebrate prey and aid colony defence.³⁷ Some examples document that negative effect of ant-derived formic acid on plant growth is not novel, but it has not been described in carnivorous plants previously. It is known that ants Myrmelachista schumanni use formic acid as a herbicide. The ants live inside the hollow stems of Duroia hirsuta, kill all plants other than their host plant by injecting formic acid into the leaves. By killing these others plants, the ants gain more nest sites and they create a single species stand of plants.³⁸ Also, weaver ants (Oecophylla smaragdina) damage mango fruit by deposition of formic acid as a result of fighting between weaver colonies.³⁹

The mechanism of action is very similar to the well known herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Formic acid causes significant inhibition of the electron transfer on the acceptor side of photosystem II, particularly from plastoquinone A to plastoquinone B.^40,41 From the data analysis of carnivorous plants published recently the genera with the highest proportions of ants in their diets are Brocchinia (90%), Nepenthes (73%) and Sarracenia (55%).⁴² All the mentioned genera have pitcher traps, with the permanent level of digestive fluid, in which the formic acid is diluted during digestion and thus the pitchers are probably prevented against its toxic effect. Captures of ants is much less frequent for sticky traps of Drosera (3.4%) and Pinguicula (0.5%); however it may have deteriorate effect.

Carnivorous plants are not just killers but are a fascinating group of plant. They do not just eat the animals but may form a complicated social network with them. Complicated animal-plant interaction, as has been described e.g. between carnivorous pitcher plant Nepenthes bicalcarata and ants may have direct impact on physiological processes similar as a well known relationship between acacia and ants.⁴³ The possible impact of formic acid of ant species co-occurring with carnivorous plants in their natural habitat on photosynthesis remains to be elucidated.

Abbreviations

A_N

rate of net photosynthesis

PAR

photosynthetic active radiation

R_D

rate of dark respiration

Φ_PSII

effective quantum yield of photochemical energy conversion in photosystem II