Photosynthetic Characterization of Australian Pitcher Plant Cephalotus Follicularis

DOI: 10.1007/s11099-011-0032-0 PHOTOSYNTHETICA 49 (2): 253-258, 2011

Photosynthetic characterization of Australian pitcher plant Cephalotus follicularis

A. PAVLOVIČ

Department of Plant Physiology, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynská dolina B2, 842 15, Bratislava, Slovakia

Abstract

Australian carnivorous pitcher plant Cephalotus follicularis Labill. produces two types of leaves. During the spring time, the plant produces a foliage type of noncarnivorous leaf called lamina. Later, the second type of leaf is produced - carnivorous pitcher. Using simultaneous measurements of gas exchange and chlorophyll (Chl) fluorescence photosynthetic efficiency of these two distinct forms of leaves were compared. In addition stomatal density, an important component of gas exchange, and Chl concentration were also determined. Pitcher trap had lower net photosynthetic rate (PN) in comparison to noncarnivorous lamina, whereas the rate of respiration (RD) was not significantly different. This was in accordance with lower stomatal density and Chl concentration in the pitcher trap. On the other hand maximum quantum yield of PSII (Fv/Fm) and effective quantum yield of photochemical energy conversion in PSII (ΦPSII) was not significantly different. Nonphotochemical quenching (NPQ) was significantly higher in the lamina at higher irradiance. These data are in accordance with hypothesis that changing the leaf shape in carnivorous plants to make it a better trap generally makes it less efficient at photosynthesis. However, the pitcher of Cephalotus had much higher PN than it was expected from the data set of the genus Nepenthes. Because it is not possible to optimize for contrasting function such as photosynthesis and carnivory, it is hypothesized that Cephalotus pitchers are less elaborated for carnivorous function than the pitchers of Nepenthes.

Additional key words: carnivorous plants; Cephalotus; chlorophyll; chlorophyll fluorescence; pitcher plants; photosynthesis; respiration; stomatal density.

Introduction

Carnivorous plants grow in nutrient poor, sunny and wet Heliamphora, Nepenthes, Sarracenia and Brocchinia, habitats. In this environment the cost of producing traps Peroutka et al. 2008, Mithöfer 2011). Transformation of is far exceeded by the benefits gained from prey capture leaves into trapping organs seriously affects their (Givnish et al. 1984). Carnivorous plants have evolved photosynthetic efficiency (Givnish et al. 1984, Adamec six times independently and represent a good example of 2006, Pavlovič et al. 2007, 2009, Karagatzides and convergent evolution (Albert et al. 1992, Ellison and Ellison 2009, Hájek and Adamec 2010). Some species Goteli 2009). Their leaves are modified into the traps, have leaves that perform photosynthesis and prey capture which attract, capture and digest an animal prey. The simultaneously (e.g. Pinguicula and Sarracenia). Other diversity of trap forms includes the famous snap-trap species are heterophyllous, with different types of leaves Venus flytrap (Dionaea muscipula), sucking bladder-trap specialized either for prey capture or photosynthesis (e.g. of bladderwort (Utricularia), fly-paper traps of, for Cephalotus and Utricularia), or with only one part of the example, sundew (Drosera) or butterwort (Pinguicula), leaf specialized for photosynthesis and the other one for and eel traps (Genlisea, Sarracenia psittacina) as well as prey capture (e.g. Nepenthes and Dionaea, Peroutka et al. pitfall-traps of pitcher plants (Cephalotus, Darlingtonia, 2008). The extent, by which the PN is reduced, may be

——— Received 13 February 2011, accepted 27 April 2011. Tel: +421 2 60296640, e-mail: [email protected] Abbreviations: F0 – minimal fluorescence in dark-adapted state; Fm – maximal fluorescence in dark-adapted state; F’m – maximal fluorescence in light-adapted state; Fv – variable fluorescence; Fv/Fm – maximal quantum yield of PSII; gs – stomatal conductance; NPQ – nonphotochemical quenching; PAR – photosynthetic active radiation; PN – net photosynthetic rate; PNmax – maximal net rate of photosynthesis at saturating irradiance; RD – rate of respiration; ΦPSII – effective quantum yield of photochemical energy conversion in PSII. Acknowledgements: This work was supported by grant VEGA 1/0040/09.

253 A. PAVLOVIČ dependent on this specialization. For example, despite leaves (Adamec 2006). In the case of D. muscipula morphological similarity, the pitchers of Sarracenia and enhanced RD is only transient as a result of generation of Darlingtonia have significantly higher PN than the action potentials (Pavlovič et al. 2010, 2011). Electrical pitchers of the genus Nepenthes with the lowest PN in the irritability may also explain seven times higher RD in pitchers (close to zero) among studied terrestrial carni- tentacles than in leaf lamina of Drosera prolifera vorous species (Ellison and Farnsworth 2005, Pavlovič et (Adamec 2010). It seems that passive pitcher plants do al. 2007, Karagatzides and Ellison 2009). It has been not usually have higher RD in their traps (Pavlovič et al. hypothesized that this discrepancy may be caused by the 2007, Adamec 2010). fact that Sarracenia has not additional organs for Cephalotus is a monotypic genus from Southwest photosynthesis and the trap must function in both; Australia with one endemic species: C. follicularis. It is a photosynthesis and prey capture to achieve a positive small, low growing terrestrial perennial carnivorous carbon gain. On the other hand the pitcher of Nepenthes pitcher plant with the heterophyllous leaves differentiated can rely on assimilates from the adjacent photosynthe- into photosynthetic lamina and the pitcher trap (Lowrie tically active lamina (Pavlovič et al. 2007). And indeed, 1998). Because photosynthetic performance in this un- recently it has been found that significant proportion of usual plant has never been studied before, gas exchange the newly fixed carbon is allocated to the trap of and Chl fluorescence were measured simultaneously in Utricularia, another genus with specialized leaves for the lamina and pitcher trap separately. Stomatal density, prey capture as well as strong reduction of photosynthesis an important component of gas exchange and Chl content in them (Adamec 2006, Sirová et al. 2010). were also determined. The results presented here indicate Greater energetic cost in traps may increase respira- that despite morphological and functional differentiation tion rate (RD) in them, especially in traps with an active of the leaves for photosynthesis (lamina) and prey capture trapping mechanism like Drosera, Dionaea and (pitcher), the pitchers had higher photosynthetic Utricularia (Adamec 2006, Adamec 2010, Hájek and efficiency than it was expected from the known data Adamec 2010, Pavlovič et al. 2010). Ion and water of the genus Nepenthes. Because the photosynthetic pumping during the resetting of Utricularia bladders is a efficiency and degree of carnivory are inversely process requiring high amounts of metabolic energy correlated, the degree of carnivory in Cephalotus in derived from respiration. And indeed RD of bladders of relation to other pitcher plants is discussed. six Utricularia species was 75–200% greater, than that in

Materials and methods

Plant culture: The endemic carnivorous pitcher plant Chl fluorescence was measured. Minimal fluorescence of –2 –1 Cephalotus follicularis Labillardiére was grown under dark-adapted state (F0 < 0.1 μmol m s PAR, 5 s) and –2 –1 greenhouse condition at Department of Plant Physiology thereafter maximal fluorescence (Fm, 4,000 μmol m s of Comenius University in Bratislava. Three-year-old, PAR, 800 ms) were measured and Fv/Fm was calculated vegetatively propagated plants were used in experiments. as (Fm – F0)/Fm. Thereafter the saturation irradiance They were grown in greenhouse in pots with peat as (1,600 μmol m–2 s–1 PAR) was applied for 15 min for substrate irrigated with distilled water at a maximum adaptation, and light response curves were recorded. The daily irradiance of 1,000 μmol m–2 s–1 PAR, day/night light was provided by blue (λ = 455 nm) and red temperature 20–30/15–20°C and high relative air (λ = 620 nm) LED diodes. PN was recorded at CO2 humidity (70–80%). Because the diameter of the cuvette concentration 360 μmol mol–1, leaf temperature 23 ± 1°C window used in gas exchange measurements was 18 mm, and relative air humidity 65–70% The light intensity was the pitchers and laminae used in the experiments did not decreased stepwise with irradiation periods of 3.5 min exceed this size. and subsequent saturation pulses (4,000 μmol m–2 s–1 PAR, 800 ms) for determination of maximal fluorescence

Simultaneous measurements of gas exchange and Chl in light-adapted state (Fm’) were applied until 60 μmol –2 –1 fluorescence: Seven laminae and seven pitchers from m s PAR was reached. Light-response curves of PN, five different plants were used for measurements. P and N ΦPSII, and NPQ were recorded simultaneously. For calcu- Chl fluorescence were measured simultaneously with a lation and definition of Chl fluorescence parameters see CIRAS-2 (PP-Systems, Hitchin, UK) and a fluorcam FC Maxwell and Johnson (2000) or Roháček (2002). After 1000-LC (Photon System Instruments, Brno, Czech the measurements the leaf tissues were dried at 70°C for Republic) attached to the infrared gas analyzer. Prior to five days, then weighed and the PN was calculated in measurements, the plants were dark-adapted for 30 min. –1 –1 nmol(CO2) g (DM) s . Thereafter the whole lamina or pitcher was enclosed in the leaf cuvette (PLC6, PP-Systems, Hitchin, UK). Once Chl extraction and quantification: Four laminae and stabilization of CO2 exchange was achieved, the rate of four pitchers were removed from four plants. Half of the respiration (RD) was measured for 5 min in the dark. Then each lamina or pitcher was dried at 70°C for 5 days to

254 PHOTOSYNTHESIS IN CEPHALOTUS PITCHER PLANT determine % of dry mass. Remaining parts of lamina or adhering onto a microscope slide. Three randomly chosen pitcher were ground in a mortar and pestle with small views of each slide were observed via an Axioskop 2 plus amount of sand and extracted with 80% (v/v) chilled microscope (Zeiss, Jena, Germany) and evaluated using acetone with MgCO3 to avoid acidification and pheophy- Laboratory Universal Computer Image Analysis software tinisation of pigments as was recommended by Ritchie (Lucia G, ver. 4.80, Laboratory Imaging, Prague, Czech (2006). The samples were centrifuged at 8,000 × g for Republic). 5 min at 4°C. Chl (a+b) in supernatant were determined spectrophotometrically (Jenway 6400, Krackeler Scien- Statistical analysis: Prior to statistical tests, data were tific, London UK): Chl a at 663.2 nm, Chl b at 646.8 nm. analysed for normality and homogeneity of variance. Chl concentration was calculated according to When nonhomogeneity was present, a t-test was Lichtenthaler (1987). employed with the appropriate corrected degrees of freedom. To evaluate the significance of the data between Determination of stomatal density: The stomatal den- lamina and pitcher trap [PN, Fv/Fm, ΦPSII, NPQ, Chl sities were determined in three laminae and pitchers using (a+b)] on the same plant individual, two-tailed paired a microrelief method. Epidermal impressions were t-test was used (Microsoft Excel). One-way ANOVA prepared by coating the leaf surface (pitcher, adaxial and followed by Least Significance Difference (LSD) test abaxial surface of lamina) with nail varnish, peeling off (Statgraphics, Centurion XV, Warrenton, Virginia,USA) the dried layer of nail varnish using sticky tape and was used to evaluate the differences in stomatal density.

Results

Fig. 1 shows differences in the leaf types (lamina and then reached saturation under an irradiance of about –2 –1 pitcher) produced by C. follicularis. RD did not differ 700 μmol m s PAR. The PNmax of pitchers was about significantly between lamina and pitcher (P=0.387, 60% that of the laminae at saturating irradiance (Fig. 2A). Fig. 2A, zero point on x-axis). The pitcher had The stomatal conductance (gs) was not evaluated, because significantly lower PN in comparison to the lamina the walls of the pitcher are wetted by digestive fluid what irrespective of light intensity. The shape of light-response did not enable the correct determination of gs. However, curves of lamina and pitcher trap was similar. First, PN high gs in the lamina indicated that stomata were open. increased almost linearly with increasing irradiance at On the other hand, ΦPSII did not differ significantly irradiances less than about 200 μmol m–2 s–1 PAR and between lamina and pitcher at any irradiance (Fig. 2B).

Fig. 1. Two types of leaves of Cephalotus follicularis: pitcher (A) and lamina (B).

At higher irradiances, NPQ was significantly higher in may be caused by the fact that the back side of the pitcher the lamina (Fig. 2C). Maximum quantum yield of PSII usually lies buried in the ground and is etiolated. Chl (Fv/Fm) was also not significantly different between leaf concentration was significantly higher in the lamina [2.51 –1 –1 types. The lamina had Fv/Fm = 0.785 ± 0.01, the pitcher ± 0.05 mg g (DM)] than in the trap [1.93 ± 0.12 mg g had Fv/Fm = 0.761 ± 0.02 (P=0.259, mean ± SE, n = 7). (DM)], P=0.034, mean ± SE, n = 4). Fig. 3 shows spatial distribution of Fv/Fm and ΦPSII. It is Fig. 4 shows stomatal density on the external surface evident that the back lower side of the pitcher had lower of pitcher (Fig. 4A), lower abaxial (Fig. 4B) and upper Fv/Fm and ΦPSII than the upper side exposed to sun. This adaxial surface (Fig. 4C). The lamina had stomata on

255 A. PAVLOVIČ upper as well as lower surface, however on the upper lower surface of the lamina than on the external surface surface they were scarce (lower surface = 58.3 ± 1.7, of the trap (11.5 ± 2.38 stomata mm–2, P<0.001, n = 9). upper surface = 3.3 ± 0.6, stomata mm–2, P<0.001, The number of stomata differs from each other (P<0.001, n = 9). In general, stomatal density was higher on the LSD test, n = 9).

Discussion

Carnivorous plants have usually low PN in comparison to Bruzzese et al. 2010, Hájek and Adamec 2010). The noncarnivorous plants. Values of PNmax determined for PNmax of Cephalotus pitcher [27.3 ± 2.5 nmol(CO2) lamina and pitcher trap of C. follicularis are within the g–1(DM) s–1] was only slightly lower than those in range as those published for numerous terrestrial Sarracenia and very similar to Darlingtonia pitchers, carnivorous species (Ellison 2006). Although the PN of however much higher than was found in pitchers of five –1 –1 the pitcher was significantly lower than in the lamina Nepenthes taxa [–4.4–2.5 nmol(CO2) g (DM) s ] by (Fig. 2A), we had expected much lower values in it. The Pavlovič et al. (2007, 2009) and Kargatzides and Ellison PN of the pitcher of different carnivorous Nepenthes (2009). Also the PN of Utricularia bladders was 7–10 species was near to zero, whereas Nepenthes lamina had times lower than in photosynthetic leaves. This indicates photosynthetic activity comparable with other carni- that the plants with two type of leaves or the part of leaf vorous species including C. follicularis (Pavlovič et al. specialized either for photosynthesis or carnivory may 2007, 2009, Karagatzides and Ellison 2009). On the other have much lower PN in their traps than species with one hand, in 10 species of American pitcher plants of the type of leaf which must both: photosynthesize and genus Sarracenia PN ranged between 29.4–72.0 capture prey. Despite this morphological specialization of –1 –1 nmol(CO2) g (DM) s and in Darlingtonia californica Cephalotus leaves, PN in the pitcher is much closer to the –1 –1 PN was 27.5 nmol(CO2) g (DM) s (Ellison and species which forms only one type of leaf (e.g. Farnsworth 2005, Karagatzides and Ellison 2009, Darlingtonia). Thus Cephalotus pitchers are probably much less dependent on assimilates produced by the lamina than for example Utricularia bladder or Nepenthes pitchers. Besides reduced PN, Cephalotus pitcher shows other characteristics related to the carnivorous syndrome. The stomatal density is significantly lower in the pitchers than in the lamina (Fig. 3). The similar trend was also found in the pitchers of two Nepenthes species (Pavlovič et al. 2007). Chl concentration is also reduced, as was previously found in Utricularia bladders (Knight 1992) and Nepenthes pitchers (Pavlovič et al. 2007, 2009). However the ΦPSII, which measures the proportion of the light absorbed by Chl associated with PSII that is used

Fig. 2. Net photosynthetic rate (PN, A), effective quantum yield of photochemical energy conversion in PSII (ΦPSII, B) and Fig. 3. Chlorophyll fluorescence imaging of leaf and trap of nonphotochemical quenching (NPQ, C) in lamina (close circle) Cephalotus follicularis. Maximum quantum yield of PSII and pitcher trap (open circle) in response to irradiance, Mean ± (Fv/Fm, upper row) and effective quantum yield of photo- SE, statistical differences between lamina and trap are denoted chemical energy conversion in PSII at 200 μmol m–2 s–1 PAR as * (P<0.05) and ** (P<0.01), two-tailed paired t-test, n = 7. (ΦPSII, lower row).

256 PHOTOSYNTHESIS IN CEPHALOTUS PITCHER PLANT

in photochemistry, is not significantly different between lamina and pitcher trap. This is in contrast with the pitcher plant of the genus Nepenthes with significantly lower Fv/Fm and ΦPSII in the pitcher. This indicates that efficiency of PSII photochemistry in Cephalotus pitcher is high, comparable with lamina. Because CO2 fixation and ΦPSII can correlate very well, the discrepancies can be caused by the changes in relative rates of CO2 fixation and competing processes such as photorespiration, nitrogen metabolism and Mehler reaction (Maxwell and Johnson 2000). In the case of Cephalotus pitchers, unequal light interception caused by three-dimensional structure of the pitchers may also contribute to this discrepancy. It seems that the pitchers of Nepenthes are better adapted for carnivorous than for assimilation function in comparison to the pitchers of Cephalotus. And indeed, some data indicate that Cephalotus pitchers represent an intermediate stage in the development of the pitchers, falling between Heliamphora and Nepenthes (Juniper et al. 1989). For example, Nepenthes and Cephalotus produce their own digestive enzymes; in Sarraceniaceae (including the genera Darlingtonia, Heliamphora and Sarracenia) the enzyme production is dubious and improbable and relies on the metabolic activity of inquilines (Jaffe et al. 1992, Adlassnig et al. 2011). However the digestive fluid of Cephalotus contains 5 times lower amount of acid proteinase than fluid samples from Nepenthes alata (Takahashi et al. 2009). Nepenthes mirabilis, N. albomarginata, and N. rafflesiana obtained 61.5, 53.8, and 61.8% of nitrogen from prey, respectively; C. follicularis only 26% (Schulze et al. 1997, Moran et al. 2001). On the other hand, C. follicularis take up much more trace elements like iron, manganese and pottasium in comparison to Sarracenia purpurea and Heliamphora nutans (Adlassnig et al. 2009). Because there must be trade-offs, it appears that it is not possible to maximize for photosynthetic and carnivorous function in the same type of leaf (Karagatzides and Ellison 2009). It seems that the extent by which the photosynthesis is reduced correlates with the ability of the leaf to be carnivorous. In Fig. 4. Stomata (arrows) on external surface of pitcher (A), this respect, the pitcher plants of the genus Nepenthes are lower abaxial (B) and upper adaxial lamina surface (C) of the best adapted to the carnivorous “lifestyle“ among C. follicularis. Bars = 100 μm. pitcher plants.

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