Acta Botanica 2-2016 - za web.indd ACTA BOT. CROAT. 75 (2), 2016 179 Acta Bot. Croat. 75 (2), 179–185, 2016 CODEN: ABCRA 25 DOI: 10.1515/botcro-2016-0036 ISSN 0365-0588 eISSN 1847-8476 Photochemical effi ciency, content of photosynthetic pigments and phenolic compounds in different pitcher parts of Sarracenia hybrids Martina Tušek1, Marcela Curman2, Marija Babić3, Mirta Tkalec3* 1 Galovec Začretski 43b, HR-49223 Sveti Križ Začretje, Croatia 2 Mokrice 72, HR-49243 Oroslavje, Croatia 3 Sveučilište u Zagrebu, Prirodoslovno-matematički fakultet, Biološki odsjek, Rooseveltov trg 6, HR-10000 Zagreb, Croatia Abstract – Sarracenia is a genus of carnivorous plants characterised by leaves modifi ed into pitchers which lure, trap and digest insects. The aim of this study was to analyse the photochemical effi ciency and contents of photosynthetic pigments and phenolic compounds in different pitcher parts – operculum, wing, pitcher- tube upper part and pitcher-tube lower part of three morphologically different Sarracenia hybrids. The photo- chemical effi ciency of the operculum and the pitcher-tube upper part was lower than that of the pitcher-tube lower part and wing, especially in hybrid B. In all hybrids, the wing had higher amount of chlorophyll a than other pitcher parts. In contrast, a higher amount of phenolic compounds, in particular anthocyanins, was mea- sured in the operculum and the pitcher-tube upper part, parts which are red-coloured and participate in luring and trapping insects. Although there were some differences among the hybrids, the results show that amount of phenolic compounds and photosynthetic pigments as well as photochemical effi ciency are related to the function of the pitcher part analysed. Keywords: anthocyanins, carnivorous plants, carotenoids, chlorophyll, fl avonoids, photosynthesis, Sarracenia * Corresponding author, e-mail: mtkalec@zg.biol.pmf.hr M. Tušek and M. Curman equally contributed to the work Introduction Sarracenia is a genus comprising 11 carnivorous spe- cies. Those rosette-forming perennials have modifi ed their leaves into pitchers, which perform a dual role – photosyn- thesis and the capture of insects (Ellison and Gotelli 2002, Pavlovič et al. 2007). Sarracenia plants attract their insect prey with extrafl oral nectaries and the pitchers’ colourful patterns (Rodenas 2012). Sarracenia species, as well as their numerous hybrids that occur in the wild and cultiva- tion, greatly differ in morphology and the colouration of pitchers (Rice 2006). The pitchers may be erect or decum- bent, yellow-green to red and pitcher-tube upper part and hood can have red veins and/or white translucent areolation (Schnell 2002). There are three types of pigments involved in the Sarracenia pitcher colouration: chlorophylls, carot- enoids and fl avonoids. The red of pitchers can be attributed to the presence of fl avonoids, in particular anthocyanins (Sheridan and Griesbach 2001, Rodenas 2012). Besides at- tracting the insects, fl avonoids have a signifi cant role in protection from high intensities of visible and UV radiation, as well as being antioxidants (Close and McArthur 2002). The main pitcher parts (Fig. 1) in Sarracenia species are the pitcher tube, pitcher mouth, nectar roll (peristome), lid (operculum) and wing (ala) (Rice 2006). The pitcher mouth is surrounded by a peristome covered with nectar-secreting glands that attract insects into the pitcher tube. Above the pitcher mouth, there is a pitcher operculum which is often coloured red and covered with glands that also secrete nec- tar and attract prey. Besides preventing rain from fi lling the pitcher tube, the pitcher lid also serves as a landing plat- form for fl ying insects (D`Amato 2013). The wing has nec- tar trails that lead insects to the pitcher mouth. The pitcher tube can be divided into upper and lower part; the upper part secretes nectar and smooth waxy material, therefore taking a role in attracting insects and preventing their es- cape (Rice 2006). In lower part of the pitcher there are glands that secrete digestive enzymes enabling digestion of prey and absorption of minerals (Glenn and Bodri 2012). Since the fi rst Darwin’s experiments, it has been as- sumed that carnivorous plants compensate for the lack of minerals in their natural habitat by capturing and digesting the insect prey. Recent studies have proven the benefi t of TUŠEK M., CURMAN M., BABIĆ M., TKALEC M. 180 ACTA BOT. CROAT. 75 (2), 2016 carnivory through increased rate of photosynthesis, bio- mass and fertility (Givnish et al. 1984, Farnsworth and El- lison 2008, Pavlovič et al. 2014). However, carnivory en- tails also some costs due to energy investment in attracting and trapping the prey (Givnish et al. 1984, Ellison and Farnsworth 2005). Namely, to catch and digest their prey, carnivorous plants have transformed some of their photo- synthetic leaves into traps with metabolically active glands, resulting in an overall decrease of photochemical effi ciency (Adamec 2010). The recent studies have shown that some Sarracenia species have different metabolic activities in wing and pitcher tube, which has more glandular tissue, suggesting that there might be also a difference in photo- synthetic activity between different pitcher parts (Adamec 2010). In the genus Darlingtonia, which is similar to the genus Sarracenia, the pitcher tube contained a higher amount of chlorophyll in regard to the hood-shaped opercu- lum, which is more reddish and has many translucent are- oles. Also, pitcher parts with extrafl oral nectaries have a lower amount of photosynthetic pigments (Ellison and Farnsworth 2005). The aim of this study was to analyse content of photo- synthetic pigments and phenolic compounds, as well as photochemical effi ciency of different pitcher parts in mor- phologically different hybrids of the genus Sarracenia. Materials and methods Morphological characteristics of hybrids The research was carried out on three morphologically different hybrids of the genus Sarracenia L., named hybrid A, hybrid B and hybrid C (Fig. 1). The plants were grown in plastic pots containing peat, and watered daily with dis- tilled water. There was always 2–3 cm of water in the plates beneath the pots. During spring and summer, the plants were grown outdoors where they were exposed to photo- synthetically active radiation and UV irradiance of maxi- mum daily levels ca. 2000 μmol photons m−2 s−1 (quantum sensor Hansatech Quantitherm, UK) and 30 W m−2 (UV meter YX-35UV, Taiwan, China), respectively. The average day/night temperature was 26/12 ± 3 °C. In all hybrids, operculum and the upper part of the pitcher tube were red-coloured with numerous red veins, while wing and the pitcher-tube lower part were green. There were some differences between hybrids – in pitcher size, operculum shape as well as in the colouration of oper- culum and pitcher-tube upper part. Pitchers of hybrid A (ca. 28 cm high) had a reduced wing. The back side of the hood- shaped operculum and upper part of tube had translucent areoles with a network of red veins. The mouth of the pitch- er was smaller than in the other two hybrids. Hybrid B (ca. 35 cm high) had undulating operculum and red veins spread all over operculum and pitcher-tube upper part. The space between the veins was more greenish, not white as in hy- brid A. The wing was larger than in hybrids A and C. Hy- brid C (ca. 23 cm high) had a more reddish operculum than the other two hybrids. The operculum was less undulating than in hybrid B. Although hybrids used in this study are of unknown origin, according to morphological features one of the parents of hybrid A is S. psittacina while one of the parents of hybrid B and C is S. purpurea. For each hybrid, three representative and morphologi- cally similar pitchers were used. Four different pitcher parts: pitcher-tube upper part, pitcher-tube lower part, wing and operculum were analysed in the experiment (Fig. 1). Photochemical effi ciency of PSII Fluorescence of chlorophyll a in vivo was measured us- ing fl uorometer (Qubit Systems Inc., Canada), with the method described by Lichtenthaler and Babani (2004) and Lichtenthaler et al. (2005). Before measurement, the pitcher samples were kept on well-watered fi lter paper in the dark for 20 minutes. The dark-adapted pitcher sample was placed on the fl uorometer stand, on wet fi lter paper, and exposed to modulated red light of low intensity (1 μmol photons m−2 s−1) to obtain the minimal fl uorescence (F0). The sample was then exposed to continuous actinic light of high inten- sity (1500 μmol photons m−2 s−1), which resulted in tran- sient increase of the fl uorescence signal from F0 to ma- ximum fl uorescence (Fm). During fi ve minutes of light exposure, the fl uorescence signal decreased, reaching steady -state fl uorescence condition (Fs). Results were recorded by software Logger Pro 3.2. Recorded data were used for cal- culation of maximum quantum yield (Fv/Fm), Fv/F0 and fl uo- rescence decrease ratio (RFd) according to Lichtenthaler et al. (2005). Photosynthetic pigments For determination of photosynthetic pigments, 50 mg of samples were homogenised with mortar and pestle in 1.5 mL of cold 80% (v/v) acetone with the addition of calcium carbonate. Homogenates were centrifuged for 10 minutes at 5000 g and 4 °C. The absorbance of supernatants was mea- sured at 470 nm, 646 nm and 663 nm using a UV/VIS spec- Fig. 1. Pitchers of three different Sarracenia hybrids (A, B and C) and their main parts used in the study. PHOTOSYNTHETIC EFFICIENCY, PIGMENTS AND PHENOLIC COMPOUNDS IN SARRACENIA ACTA BOT. CROAT. 75 (2), 2016 181 trophotometer Specord (Analytik Jena). The content of chlo- rophyll a, chlorophyll b and total carotenoids was de termined according to Lichtenthaler (1987) and Wellburn (1994). Total phenolics, fl avonoids and anthocyanins Pitcher samples (50 mg) were homogenised with mortar and pestle in 1.5 mL of cold 50% (v/v) ethanol. After incu- bation at 60 °C for 30 minutes, the homogenates were cen- trifuged at 12000 g for 10 minutes. The supernatants were used to measure total phenolics, fl avonoids and anthocya- nins. For total phenolics content, a mixture of 1580 μL of dis- tilled water, 20 μL of plant extract, 100 μL of Folin–Ciocal- teu reagent and 300 μL of 1.88 M sodium carbonate was prepared. After incubation at 45 °C for 60 minutes, the ab- sorbance at 765 nm was measured using a UV/VIS spectro- photometer (Singleton et al. 1999). The results were ex- pressed as mg of gallic acid equivalents (GAE) per g of fresh weight. For total fl avonoids content, an aliquot of the plant ex- tract (100 μL) was mixed with 20 μL of 10% (w/v) AlCl3, 500 μL of 1 M potassium acetate and 380 μL of distilled water. The mixture was incubated at 24 °C for 30 minutes and absorbance at 420 nm was measured using a UV/VIS spectrophotometer (Pourmorad et al. 2006). The results were expressed as mg of quercetin equivalents (QE) per g of fresh weight. For total anthocyanins content, 500 μL of plant extract was mixed with 500 μL of 50% (v/v) ethanol and 84 μL of 37% HCl. After incubation at 60 °C for 30 minutes, the ab- sorbance was measured at 530 nm using a UV/VIS spectro- photometer (Paiva et al. 2003). Total anthocyanin content was determined using a molar extinction coeffi cient of 34300 M–1 cm–1 and a molecular weight of 449.2 g mol–1, and expressed as mg of cyanidin-3-glucoside equivalents (C3GE) per g of fresh weight. Statistical analysis Results were shown as means ± standard errors. Deter- mination of phenolic compounds and photosynthetic pig- ments content was performed in six replicates. Determina- tion of photochemical effi ciency was performed in triplicate. For processing data Microsoft Excel 2007 and Statistica 10 (StatSoft Inc., SAD) were used. The results obtained for different pitcher parts within each hybrid were compared by analysis of variance (ANOVA) and post hoc Tukey’s test. Differences between means were considered statistically signifi cant at p ≤ 0.05. Results Photochemical effi ciency of PSII All pitcher parts of all hybrids had a similar maximum quantum yield of PSII (Fv/Fm) with values ranging from 0.72 to 0.78 (Tab. 1). The exceptions were much lower Fv/ Fm values noticed in the pitcher-tube upper part and opercu- lum of hybrid B, the operculum showing an extremely low value (0.37). The highest value of Fv/F0 was detected in the wing of all hybrids investigated, while the lowest value was found in the operculum. Analysis of pitcher parts of hybrid A showed a signifi cant decrease of Fv/F0 in the pitcher-tube upper part and operculum compared to the wing. Amongst hybrids, hybrid B stands out with the lowest Fv/F0 values measured in the operculum and the pitcher-tube upper part. In hybrid C, there was no signifi cant difference in Fv/F0 be- tween pitcher parts, though the highest value was observed in the wing (Tab. 1). In hybrid A, there was no signifi cant difference in RFd values among different pitcher parts although a slight de- crease was determined in the pitcher-tube lower part. In hy- brid B, the highest and the lowest RFd values were measured in the wing and operculum, respectively. The operculum of hybrid B had the lowest RFd value. There was no signifi cant difference in RFd values among the different pitcher parts in hybrid C (Tab. 1). Content of photosynthetic pigments In all three hybrids, the chlorophyll a content was sig- nifi cantly higher in the wing than in the other parts of pitch- ers. The wing of hybrid B had the highest amount of chloro- phyll a. A high chlorophyll a content was also detected in the pitcher-tube lower part of hybrid B. In hybrids A and C, chlorophyll a contents in the pitcher-tube upper and lower part as well as operculum did not differ from each other (Fig. 2A). In hybrids B and C, the highest amounts of chlorophyll b and total carotenoids were detected in the wing while in hybrid A the highest amount was detected in the operculum. Tab 1. Chlorophyll a fl uorescence parameters: maximum quan- tum yield of PSII (Fv/Fm), ratio between variable and minimum fl uorescence (Fv/F0) and chlorophyll fl uorescence decrease ratio (RFd), in three different Sarracenia hybrids (A, B and C). Four pitcher parts: pitcher-tube upper part (PU), pitcher-tube lower part (PL), wing (W) and operculum (O) were analysed. The results were expressed as the average of 3 replicates ± standard error. Dif- ferent letters in each column denote signifi cantly different results (p ≤ 0.05) among different pitcher parts within each hybrid, ns – not signifi cant. Sarracenia hybrids Pitcher part Fluorescence parameters Fv/Fm Fv/F0 RFd A PU 0.74±0.01ab 2.86±0.12a 2.83±0.09ns PL 0.77±0.01ab 3.30±0.14ab 2.15±0.22ns W 0.78±0.003b 3.62±0.06b 2.66±0.33ns O 0.72±0.02a 2.63±0.25a 2.72±0.22ns B PU 0.57±0.07b 1.41±0.34a 1.88±0.44ab PL 0.77±0.003c 3.36±0.05b 2.43±0.40ab W 0.78±0.001c 3.59±0.02b 2.94±0.09b O 0.37±0.04a 0.61±0.11a 1.18±0.19a C PU 0.75±0.01ns 3.02±0.20ns 3.21±0.07ns PL 0.73±0.02ns 2.77±0.25ns 2.83±0.33ns W 0.77±0.01ns 3.37±0.13ns 3.06±0.21ns O 0.74±0.02ns 2.81±0.24ns 3.76±0.32ns TUŠEK M., CURMAN M., BABIĆ M., TKALEC M. 182 ACTA BOT. CROAT. 75 (2), 2016 The wing of hybrid B had the highest amount of chloro- phyll b and carotenoids. The lowest contents of chlorophyll b and carotenoids were observed in the pitcher-tube lower part of hybrid A, as well as in the pitcher-tube lower and upper part of hybrid C when compared to the wing and operculum (Figs. 2B and C). In hybrids A and B the chlorophyll a/b ratio and the chlorophylls/carotenoids ratio were higher in the wing and pitcher-tube lower part than in the other two pitcher parts. In hybrid C the highest values of both ratios were observed in the wing, while the operculum had the lowest chloro- phyll a/b ratio (Tab. 2). Content of phenolic compounds Among different pitcher parts, the highest amount of to- tal phenolics was detected in the operculum, especially in hybrids A and C. In hybrids A and B, the pitcher-tube lower part and the wing had the lowest amount of total phenolics, while in hybrid C the lowest amount was measured in the pitcher-tube upper part (Fig. 3A). In all analysed hybrids, the operculum showed the high- est and the pitcher-tube lower part the lowest content of fl a- vonoids. In hybrid C, the wing also contained a higher amount of fl avonoids than the pitcher-tube upper and lower part (Fig. 3B). The operculum was the pitcher part with the highest amount of anthocyanins, especially in hybrid A. In hybrids B and C, the pitcher-tube upper part also contained a high amount of anthocyanins. In all hybrids, the pitcher-tube lower part and the wing had lower anthocyanin content than the other pitcher parts (Fig. 3C). Discussion The optimal effi ciency of PSII (Fv/Fm) is an important indicator of photochemical effi ciency in plants (Maxwell and Johnson 2000). In all the hybrids investigated, the mea- sured Fv/Fm values (Tab. 1) were in accordance with theore- tical values (0.74 to 0.85) for non-carnivorous plants (Max- well and Johnson 2000, Lichtenthaler et al. 2005). However, Fv/F0, a more sensitive fl uorescence parameter (Lichtentha- ler et al. 2005), showed values below the critical value of 3.8, indicating a decrease of photochemical effi ciency (Cha- tzistathis et al. 2011) in all pitchers parts (Tab. 1). A lower photochemical effi ciency has been often found in carnivo- rous plants (Bruzzese et al. 2010) and it has been correlated with the costs of carnivory (Adamec 2010) as well as with low foliar nitrogen content and slow growth of carnivorous plants (Ellison and Adamec 2011, Pavlovič and Saganová 2015). The lowest values of both parameters, Fv/Fm and Fv/F0 were found mostly in the operculum and upper part of the pitcher tube, which are predominantly red-coloured areas with numerous nectar-secreting glands. Moreover, in these pitcher parts a lower amount of chlorophyll a and higher Tab 2. Chlorophyll a/ b ratio (chl a/b) and chlorophylls/carot- enoids ratio (chl/car) in three different Sarracenia hybrids (A, B and C). Four pitcher parts: pitcher-tube upper part (PU), pitcher- tube lower part (PL), wing (W) and operculum (O) were analysed. The results were expressed as the average of 6 replicates ± stan- dard error. Different letters in each column denote signifi cantly different results (p ≤ 0.05) among different pitcher parts within each hybrid. Sarracenia hybrids Pitcher part chl a/b chl/car A PU 2.12±0.38ab 2.11±0.12b PL 2.75±0.06b 2.47±0.06bc W 2.98±0.08b 2.54±0.04c O 1.75±0.28a 1.74±0.12a B PU 1.91±0.17a 1.83±0.12a PL 2.44±0.06b 2.33±0.12b W 2.37±0.1b 2.9±0.13c O 1.86±0.08a 1.49±0.11a C PU 2.35±0.11b 2.11±0.12ab PL 2.28±0.16ab 1.82±0.06a W 2.58±0.15b 2.45±0.09b O 1.8±0.17a 1.89±0.1a A B C a a b a ab b c a a a b a 0 0.1 0.2 0.3 0.4 0.5 0.6 PU PL W O PU PL W O PU PL W O Hybrid A Hybrid B Hybrid C C on te nt o f c hl or op hy ll a (m g g F W ) ab a ab b a a b a a a b b 0 0.05 0.1 0.15 0.2 0.25 0.3 PU PL W O PU PL W O PU PL W O Hybrid A Hybrid B Hybrid C C on te nt o f c hl or op hy ll b (m g g F W ) ab a bc c a a b a a a b b 0 0.05 0.1 0.15 0.2 0.25 0.3 PU PL W O PU PL W O PU PL W O Hybrid A Hybrid B Hybrid C C on te nt o f t ot al c ar ot en oi ds (m g g F W ) Fig. 2. Content of chlorophyll a (A), chlorophyll b (B) and total carotenoids (C) in three different Sarracenia hybrids. Four pitcher parts: pitcher-tube upper part (PU) and lower part (PL), wing (W) and operculum (O) were analyzed. The results were expressed as the average of 6 replicates ± standard error. Different letters above the bars denote signifi cantly different results (p ≤ 0.05) among dif- ferent pitcher parts within each hybrid. FW – fresh weight. PHOTOSYNTHETIC EFFICIENCY, PIGMENTS AND PHENOLIC COMPOUNDS IN SARRACENIA ACTA BOT. CROAT. 75 (2), 2016 183 amount of phenolic compounds were found (Figs. 2 and 3), which is in accordance with higher anthocyanin and lower chlorophyll content found in the traps of Nepenthes (Pavlovič and Saganová 2015). On the other hand, the high- est photochemical effi ciency and chlorophyll content were recorded in the green-coloured wing of Sarracenia, a pitch- er part that has a role in guiding insects to the pitcher mouth (D`Amato 2013). However, the lower part of the pitcher tube, although green-coloured, had lower photochemical effi ciency, probably due to the presence of digestive glands needed for the digestion and absorption of minerals (D`Amato 2013). The lower Fv/Fm values and photosynthe- sis rate in the trapping organs of carnivorous plants other than Sarracenia have already been described (Pav lovič et al. 2009). We also measured the fl uorescence decrease ratio (RFd), a parameter directly related to rate of photosynthesis (Lichtenthaler and Babani 2004). The measured RFd values (Tab. 1) were mostly between those of sun-exposed leaves (3 to 5) and those of shade-exposed leaves (1 to 2.5). High- er RFd values in sun-exposed leaves refl ect their higher pho- tosynthetic capacity and CO2 fi xation rate (Lichtenthaler et al. 2005). Carnivorous plants grow mostly in sunny habitats (Zamora et al. 1998) suggesting that RFd values should be in the range for sun-exposed plants. However, as already men- tioned, carnivorous plants mostly have a reduced rate of photosynthesis due to low foliar nitrogen content and nitro- gen incorporation into other than photosynthesis-related molecules (chlorophylls, proteins), such as those involved in adjustments for trapping and digesting insects (Ellison and Adamec 2011, Pavlovič and Saganová 2015). Unex- pectedly, in hybrid A and C high RFd values were observed not only for the green-coloured wings but also for red-co- loured pitcher-tube upper part and operculum. The wing of all hybrids investigated had signifi cantly higher content of chlorophyll a, than the operculum and pitcher tube (Fig. 2A). In the genus Sarracenia, red-co- loured veins with nectar-producing glands (Newell and Nastase 1998) are mostly distributed in the area of opercu- lum, pitcher upper part and peristome (Płachno 2007). Re- placement of cells that contain chlorophyll with glands that have an important role in the attraction and digestion of in- sects is considered to be one of the costs of carnivory (Hájek and Adamec 2010). In the genus Darlingtonia, it has been found that pitcher parts that contain more extrafl oral nectaries have a lower amount of photosynthetic pigments (Ellison and Farnsworth 2005). Unlike chlorophyll a, high values of chlorophyll b and carotenoids were found also in the operculum, especially in hybrids A and C (Fig. 2B) which coincided with the high phenolic content (especially anthocyanins, Figs. 3A and C). As anthocyanins could ab- sorb some part of photosynthetically active radiation (Gould et al. 2010), we hypothesize that increased content of chlorophyll b and carotenoids could be involved in spec- tral broadening of light absorbed by the operculum. The relatively high photosynthetic rates observed in hybrids A and C support the idea. Additionally, carotenoids could also have a role in protection from high light stress (Ramel et al. 2012) as the operculum is the pitcher part most exposed to the sunlight. The lower chlorophyll a/b ratio found in the operculum may be considered an enlargement of the anten- na system of photosystem II while a lower chlorophylls/ca- rotenoids ratio is a typical characteristic of sun-exposed leaves (Lichtenthaler and Buschmann 2001). Similar re- sults were reported by Tkalec et al. (2015) who observed a higher content of anthocyanins as well as a lower chloro- phylls/carotenoids ratio in sun-exposed Drosera rotundifo- lia and a lower chlorophyll a/b ratio in plants growing in low-light conditions. The highest content of total phenolic compounds and fl avonoids found in the operculum of Sarracenia hybrids is probably related to the presence of red-coloured phenolic compounds, like anthocyanins (Sheridan and Griesbach 2001). The operculum is variegated with a lot of red veins that have an important role in luring insects (Newell and Nastase 1998). Furthermore, phenolic compounds with their antioxidant potential can protect the operculum, the pitcher part most exposed to sunlight, from light-induced formation of free radicals (Close and McArthur 2002). In- terestingly, in all hybrids the lower part of pitcher tube had A B C a a a b bc a a c b a a c 0 0.3 0.6 0.9 1.2 1.5 PU PL W O PU PL W O PU PL W O Hybrid A Hybrid B Hybrid C C on te nt o f a nt ho cy an in s ( m g E -C 3G g F W ) a a a b b a b c a a b b 0 3 6 9 12 PU PL W O PU PL W O PU PL W O Hybrid A Hybrid B Hybrid C C on te nt o f f la vo no id s (m g E -K g F W ) b a ab c ab a a b a ab ab b 0 5 10 15 20 25 PU PL W O PU PL W O PU PL W O Hybrid A Hybrid B Hybrid C C on te nt o f t ot al p he no lic s (m g E -G A g F W ) Fig. 3. Content of total phenolics (A), fl avonoids (B) and antho- cyanins (C) in three different Sarracenia hybrids. Four pitcher parts: pitcher-tube upper part (PU) and lower part (PL), wing (W) and operculum (O) were analyzed. The results were expressed as the average of 6 replicates ± standard error. Different letters above the bars denote signifi cantly different results (p ≤ 0.05) among dif- ferent pitcher parts within each hybrid. FW – fresh weight. TUŠEK M., CURMAN M., BABIĆ M., TKALEC M. 184 ACTA BOT. CROAT. 75 (2), 2016 the lowest content of fl avonoids, probably because its func- tion is not luring the prey, but digestion of pray trapped in the tube (Glenn and Bodri 2012). By contrast, the content of fl avonoids was relatively high in the wing, the green-co- loured pitcher part with the highest content of chlorophyll a and Fv/F0. It is possible that fl avonoids protect photosyn- thetically active tissue from oxidative stress, due to their antioxidant potential and free radical quenching ability (Ba- nasiuk et al. 2012). As already mentioned, anthocyanins are known to be responsible for the red colouration of Sarrace- nia pitchers, so the highest content could be expected to be found in the operculum (Fig. 3C), the most red-coloured part of the pitcher. Besides the operculum, the upper pitcher part had also high content of anthocyanins, pointing to its role in the attraction of insects (Rice 2006). It has been found that some species of the genera Drosera and Dionaea exposed to mineral defi ciency conditions, especially nitro- gen, can increase the intensity of red colour to attract more prey (Ischiishi et al. 1999, Gao et al. 2015). The lowest an- thocyanin content was observed in the operculum of hybrid B which had a more greenish tissue between the veins. In- terestingly, the operculum of hybrid A which had translu- cent aureoles showed the highest content of anthocyanins (Figs. 1 and 3C). It is possible that leucoanthocyanins, which are not coloured, contribute to the high content of anthocyanins, but it is yet to be determined. In conclusion, our results show that variances in photo- chemical effi ciency, content of photosynthetic pigments and phenolic compounds correlate with the function of pitcher part analysed. In all Sarracenia hybrids investigat- ed, the green-coloured wing had the highest content of chlorophyll a and photochemical effi ciency which is con- nected with its role in photosynthesis. The red-coloured operculum and upper pitcher part, whose role is luring and catching prey, contained more phenolic compounds, espe- cially anthocyanins, which help to attract insects. References Adamec, L., 2010: Dark respiration of leaves and traps of terres- trial carnivorous plants: are there greater energetic costs in traps? Central European Journal of Biology 5, 121–124. 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