Photosynthesis and NitrogenMetabolism of Nepenthes alata in Response to Inorganic NO 3 − and Organic Prey N in the Greenhouse

This study investigates the relative importance of leaf carnivory on Nepenthes alata by studying the effect of different nitrogen (N) sources on its photosynthesis and N metabolism in the greenhouse. Plants were given either inorganic NO3 −, organic N derived from meal worms, Tenebrio molitor, or both NO3 − and organic N for a period of four weeks. Leaf lamina (defined as leaves) had significant higher photosynthetic pigments and light saturation for photosynthesis compared to that of modified leaves (defined as pitchers). Maximal light saturated photosynthetic rates (Pmax) were higher in leaves than in pitchers. Leaves also had a higher light utilization than that of pitchers. Both leaves and pitchers of plants that were supplied with both inorganic NO3 − and organic prey N had a similar photosynthetic capacity and N metabolism compared to plants that were given only inorganic NO3 −. However, adding organic prey N to the pitchers enhanced both photosynthetic capacity and N metabolism when plants were grown under NO3 − deprivation condition. These findings suggest that organic prey N is essential for N. alata to achieve higher photosynthetic capacity and N metabolism only when plants are subjected to an environment where inorganic N is scarce.


Introduction
Carnivorous plants are restricted to environments with an abundant supply of water and light but are poor in nutrients [1].Although plants are autotrophic with respect to reduced carbon, they must scavenge nitrogen (N) and other minerals from the environment, usually from the soil through uptake by their roots.On the other hand, plant carnivory is an alternate and efficient means to acquire nutrients in nutrientpoor habitats [2].For instance, preys caught in the Nepenthes pitchers are digested in a pool of digestive enzymes in the pitcher where glands function to perceive chemical stimuli, secrete digestive enzymes, and absorb nutrients for plant growth and development [3].
Nepenthes are tropical pitcher plants, and there are approximately 90 species in the genus Nepenthes.Osunkoya et al. [4] suggested that most Nepenthes species are N-(but not P or K) limited, and thus have evolved the pitcher to assist in their uptake of N. The leaf morphology of the different Nepenthes species is similar with a photosynthetic lamina and a tendril to which a pitcher is attached.N. alata is a pitcher plant that efficiently captures, retains, and digests predominantly insect prey in highly modified leaves, and pitchers [5].The pitcher consists of the lid, peristome (upper rim of pitcher) which attracts prey, a waxy zone that is involved in trapping prey, and a digestive zone which digests prey [6].Ellison and Gotelli [2] reported that both leaves and pitchers of the Nepenthes plants can photosynthesize.Clarke [7] found that Nepenthes fail to produce pitchers if the light or humidity is too low, or nutrient availability is too high.
In Singapore, N. alata is a popular ornamental plant that can be found in many home gardens and has commercial value.In nature, these large pitcher plants usually grow in soils consisting of low N, as the pitchers of these plants can obtain N from organic sources like insects or small animals.Usually, N. alata plantlets are obtained from tissue culture stock in the nurseries.Some growers of Nepenthes feed the pitchers of this plant with meal worms to provide the additional source of N.However, there are only a few studies that have examined directly the linkage between inorganic N uptake from the soil by the carnivorous plants and photosynthetic rate [5].In addition, there is little information available on the overall prey and inorganic nutrient acquisition for the carnivorous plants such as Nepenthes [5].
This project focused on the relative importance of leaf carnivory and root nutrition mainly with inorganic NO 3 − on photosynthesis and N metabolism of N. alata in the greenhouse.Using NO 3 − and prey-derived organic N source, this project aimed to compare the photosynthetic characteristics and light utilization between leaf and pitcher and to study the effects of NO 3 − and prey on the photosynthesis and N metabolism of leaf and pitcher.The parameters studied were photosynthetic O 2 evolution, chlorophyll (Chl) fluorescence, photosynthetic pigments, total reduced N content, and soluble protein content.Understanding the contributions of inorganic NO 3 − and organic prey N to photosynthesis and N metabolism, horticulturalists can select the optimal fertilizer required for cultivation of N. alata.

Materials and Methods
2.1.Plant Material.N. alata plants with 7-8 leaves and 4-5 pitchers were obtained from a commercial nursery.They were transplanted to pots (15 cm diameter) containing sand and vermiculite (1 : 1), and each pot had only one plant.The pitchers were emptied and washed with distilled water.An amount of 10 mL of distilled water was added to each of the pitchers which were then plugged with glass wool to prevent colonization by common pitcher inhabitants and capture of prey.All plants were acclimatized for one month in the greenhouse under a maximal photosynthetic photon flux density (PPFD) of 600-700 µmol m −2 s −1 .The daily ambient temperature ranged from 24 to 33 • C. All plants were watered daily with tap water and supplied with nutrient solution based on full-strength Netherlands Standard Composition every alternate day.This nutrient solution contains full NO 3 − .

Experimental Design for Different N Treatments.
After the plants were acclimatized under the previously stated conditions for one month, all yellow leaves and dead pitchers were removed.Each plant had 6 fully expanded leaves with fully developed pitchers and two young leaves without pitchers.The pitchers were emptied and washed with distilled water.An amount of 10 mL of distilled water was again added to each of the pitchers which were then plugged with glass wool to prevent colonization by common pitcher inhabitants and capture of prey.In order to study the photosynthetic characteristics and N metabolism in response to organic prey and inorganic NO 3 − , for each plant, six alive meal worms (Tenebrio molitor) (6 × 0.4 g) were added to each of the three pitchers, while the other three pitchers of the same plant did not receive any prey.After adding the meal worms, plants were divided into two groups: one group was watered nutrient solution with full NO 3 − , while the other group was watered with nutrient solution without NO 3 − every alternate day.Therefore, there are four treatments: (1) NO 3 − , (2) NO 3 − + prey, (3) prey, and (4) no NO 3 − , no prey.The durations of different treatments were two, three, and four weeks, respectively.Significant differences in responses to different treatments were observed after four weeks.Thus, only data obtained after four weeks were presented.

Measurement of Chl
Fluorescence.Electron transport rate (ETR), photochemical quenching (qP), and nonphotochemical quenching (qN) of Chl fluorescence were determined from both leaves and pitchers using the Imaging PAM Chl Fluorometer (Waltz, Effeltrich, Germany) at 25 • C under different PPFDs in the laboratory as described by He et al. [8].

Measurement of Photosynthetic O 2
Evolution.The photosynthetic O 2 evolution of leaf and pitcher were determined with a Hansatech leaf disc O 2 electrode (King's Lynn, Norfolk, UK).Each leaf and pitcher section was placed in saturating CO 2 conditions (1% CO 2 from 1 M carbonate/bicarbonate buffer, pH 9).Leaf or pitcher section was illuminated, starting from the lowest photosynthetic photon flux density, PPFD (34 µmol m −2 s −1 ), to the highest (1000 µmol m −2 s −1 ).The photosynthetic light response curve was obtained by plotting the O 2 evolution rates against respective light intensity.Maximal photosynthetic O 2 evolution rates (P max ) of both leaf and pitcher were measured after two weeks of treatments under a PPFD of 1000 µmol m −2 s −1 at 25 • C.

Measurement of Photosynthetic Pigments.
Fresh samples of leaf or pitcher of 0.05 g were weighed and cut into smaller pieces.Total Chl and carotenoid were extracted from these samples with dimethylformamide and quantified using a spectrophometer following the procedure of Wellburn [9] at wavelengths of 480, 647, and 664 nm.

Measurement of Total Reduced N Concentration (TRN).
Dry samples of 0.05 g of leaf and pitcher were placed into a digestion tube with a Kjeldahl tablet and 5 mL of concentrated sulphuric acid according to Allen [10].The mixture was then digested about 60 min until clear.After the digestion was completed, the mixture was allowed to cool for 30 min, and TRN concentration was determined by a Kjeltec 2030 analyser unit (Höganäs, Sweden).

Total Soluble Protein (TSP) Extraction and Determination.
Samples of 1 g were rapidly frozen in liquid nitrogen after weighing and stored at −80 • C until used.Each leaf and pitcher sample was ground to fine powder in liquid N with pestle and mortar.After which, 1 mL of 100 mM Bicine-KOH (pH 8.1), 20 mM MgCl 2 , and 2% PVP buffer were added [11].After centrifugation (100,000 g, 30 min at 4 • C), 4 mL of acetone was then added to 1 mL of the supernatant collected and centrifuged further for 10 min at 4000 rpm.Total soluble protein was extracted using the method described by Lowry et al. [11].

Statistical Analysis.
For Table 1 and Figures 1 and 2, a t-test was used to test for differences between leaves and

Comparative Studies on Photosynthetic Characteristics between Leaves and Pitchers.
To compare the photosynthetic characteristics between leaves and pitchers, all plants were grown under the conditions described in Section 2.1 for one month.The leaves had significant higher total Chl content, Chl a/b ratio, and total carotenoid content compared to that of the pitchers (Table 1, P < 0.05).However, there was no significant difference in the Chl/carotenoid ratio between leaves and the pitcher.Light saturation point of photosynthetic O 2 evolution for leaf was achieved at PPFDs of about 600-800 µmol m −2 s −1 (Figure 1).For the pitchers, however, light saturation point was much lower, at PPFDs of about 200-400 µmol m −2 s −1 .These results indicate that the leaves have higher photosynthetic capacities compared to those of pitchers.Light utilization of leaf and pitcher was determined by qP, qN, and ETR.The leaves had qP values of about 0.9 to 0. On the other hand, the leaves had a gradual increase in qN from PPFD of 25-400 µmol m −2 s −1 after which it plateaued to about 0.8.Similarly, the pitcher had a rapid increase in qN from PPFD of 25-400 µmol m −2 s −1 after which it plateaued to about 0.6 (Figure 2(a)).Compared to that of pitcher, the higher qN levels in leaves indicate that higher amount of light energy could be dissipated as heat.− to the roots and prey added to the pitcher (Figures 3(a) and 3(c)).However, leaves that were supplied with prey to their pitchers only but without NO 3 − to the roots had significant lower P max and Chl content (P < 0.05).Leaves that had neither NO 3 − supplied to the roots nor prey added to the pitchers exhibited the lowest P max and Chl content (Figures 3(a) and 3(c), P < 0.05).After different N treatments, the changes of P max in pitchers were very similar to those of leaves but the values of P max were much lower compared to those of leaves (Figure 3(b)).However, lower total Chl content was only observed in pitchers of those plants without NO 3 − and prey (Figure 3(d), P < 0.05).Changes in TRN and TSP determined from the same leaves that were used to measure P max and total Chl content are shown in Figure 4.The differences in TRN concentrations of both leaves and pitchers were very similar to those of P max after different N treatments for 4 weeks (Figures 4(a

Discussion
Carnivorous plants normally grow in moist, nutrient-poor soils.In order to adapt to an environment where critical nutrients are scarce and where light is not limiting, carnivorous plants have evolved modified leaves specialized for capturing animals and digesting the preys to acquire nutrients [1,[12][13][14][15].In this study, using the carnivorous tropical pitcher plant, N. alata, it was demonstrated that the leaves have much higher P max compared to that of the pitchers of the same plants (Figure 1).It was also reported by others that P max of traps is usually lower than that of other noncarnivorous leaves of the same plants [1].This could be due to the fact that leaves have higher levels of photosynthetic pigments compared to the pitcher (Table 1) and high efficiency of light energy utilisation and heat dissipation measured by qP, qN, and ETR (Figure 2).Pavlovič et al. [16] also reported that Chl content in two Nepenthes species, N. alata and N. mirabilis, was higher in the lamina (leaves) than in the pitcher.The red tint of Nepenthes pitchers suggests that they might not have much Chl, and this might lead to low P max .
Because P max is positively correlated with N concentration and stomatal conductance, it is hypothesized that there is lower N concentration (Figure 4) and lower stomata density in pitchers than in the leaves.Most carnivorous plants exhibit very low rates of photosynthesis [5].In the present study, although P max was significantly higher in leaves than in pitchers, the value of P max about 10 µmol m −2 s −1 was much lower compared to that of most C 3 plants.According to Ellison [5], photosynthetic rate of carnivorous plants was about 2 to 5 times lower than that of other noncarnivorous plants.Our finding of P max of N. alata agrees with Ellison's report [5].Low photosynthetic rate reflects the relatively low growth rate of N. alata plants (data not shown).Most carnivorous plants are at a competitive disadvantage in their habitats due to a lower net photosynthetic rate when light is limiting, and the availability of soil nutrients is poor based on the cost-benefit model [1].However, the relationship between photosynthetic performance of carnivorous plants and their carnivory is complex and ambiguous [3].
According to the ecological cost-benefit relationships, carnivory of carnivorous plants grown under their natural habitat of limited light and poor nutrients could lead to increasing photosynthetic rate if they were provided with a greater mineral nutrient availability [1].N. alata is one of the most popular Nepenthes species in cultivation.According to our observation, N. alata plants that are cultivated in the local nursery under high light supplied with fertilizer grow well with numerous pitchers.These observations lead to the question of the relative importance of leaf carnivory and root nutrition mainly with inorganic NO 3 − on photosynthesis and N metabolism.In the present study, when full inorganic NO 3 − was supplied to the roots of N. alata plants, adding preys to the pitchers did not increase P max and total Chl content of both leaves and pitcher (Figure 3).These results indicate that to improve plant growth, it seems sufficient to provide N in the form NO 3 − to the roots.This confirms the idea that carnivory is not indispensable for greenhouse growing carnivorous plants, but it is almost indispensable for carnivorous plants in natural habitats [17].Prey captured in the pitcher could contribute 10-90% of the N budget of Nepenthes plants [16].N in the end can be considered as limiting primary productivity [18].For instance, when N. alata plants were grown under NO 3 − deprivation condition, adding preys to the pitchers increased P max of both leaves and pitchers compared to those without preys.However, the  P max values of plants supplied with only preys were significantly lower than those plants supplied with NO 3 − or both NO 3 − and preys (Figure 3).These findings suggest that in the event of low inorganic NO 3 − availability, the feeding of prey to the pitchers would bring about the same positive effect on growth (Figure 3).Chandler and Anderson [19] reported greater absolute growth in the presence of insects at low NO 3 − concentration in Drosera whittakeri and Drosera binata over a period of one growing season.In most Sarracenia species and in Darlingtonia californica, prey addition significantly increases P max [1].Ellison and Gotelli [20] concluded that there was an increase in photosynthetic rates followed by an addition of organic N in Sarracenia purpurea.The result is also consistent with another study conducted by Wakefield et al. [21] on Sarracenia purpurea.However, benefits of carnivory including an increased rate of photosynthesis are often conflicting [20][21][22].Obviously, the photosynthetic effect of prey addition is quite different in different carnivorous plant species [20][21][22][23][24][25].It was reported that the total Chl content of the younger Sarracenia pitchers was positively and significantly correlated with the feeding level of prey.However, Chl content in Sarracenia was not correlated with either foliar N [23].In the present study, even with additional prey N, leaves of N. alata plants had much higher P max and total Chl content compared to their pitchers; feeding preys to the pitcher of plants that  were supplied with full NO 3 − did not enhance Chl further (Figures 3(c) and 3(d)).These results may suggest that the function of the pitchers is mainly for prey capture and not for photosynthesis, although they have photosynthetic pigments [2].This could therefore explain why the leaves utilize light much more efficiently compared to the pitchers as demonstrated by the higher ETR, qP, and higher qN values of the leaves compared to that of the pitcher (Figure 2, Table 1).
It is well known that in noncarnivorous plants, with increasing inorganic NO 3 − content, the photosynthetic capacity of leaves increases [26,27].However, there is very little information on the relationship between the amount of organic N (prey), the rate of photosynthesis, and N metabolism in carnivorous plants.Typically, a reduction of TRN concentration in plants would lead to lower photosynthetic rates and vice versa.In this study, TRN was significantly higher in leaves than in pitchers (Figure 4).This could further explain why leaves had higher photosynthetic capacity and photosynthetic pigments compared to pitchers.In addition, differences in TRN concentrations of both leaves and pitchers among the different N-treated plants were very similar to those of P max , indicating the close relationship between TRN and P max .N availability obviously directly affects the amount of soluble proteins available in the plant, since N is a major element which makes up protein compounds [26,27].Plants require N (in the form of NO 3 − ) to synthesize soluble proteins which in turn is required for the synthesis of RuBisCo that is the key enzyme in photosynthetic CO 2 fixation [27,28].This could explain why leaves, which had significantly higher TSP compared to the pitcher (Figure 4), had higher photosynthetic capacities (Figure 2).However, how much of the prey contributes to the N actually present in the foliage of Nepenthes?Studies like Ellison and Gotelli [20] suggest that in the field, the majority of foliar N is derived from substrate sources rather than carnivory.There are other conflicting findings like Moran et al. [18] who estimated that prey contributed from 54% to 68% of the total foliar N in Nepenthes.Schulze et al. [29] concluded that the total N concentration in leaves of Dionaea muscipula growing in areas where there was little insect availability was much lower than in plants which received a large amount of insect prey.When measuring NO 3 − reductase (NR) activity, it was shown that negative interactions can exist between organic and inorganic N sources.This was exhibited when insect feeding of greenhouse Drosera binata grown in complete solution led to 30-50% lower shoot NR activity compared to unfed plants [18].Effects of organic prey on NR activity merit our future study.

Conclusion
The present study showed that the leaves of N. alata had higher photosynthetic capacity and light utilization compared to the pitchers.N. alata plants with matured pitchers supplied with both inorganic NO 3 − and organic prey N had a similar P max compared to plants supplied with only inorganic NO 3 − , suggesting that carnivory is not indispensable for greenhouse growing carnivorous plants.
6 under PPFD of 15-200 µmol m −2 s −1 .A drastic decrease of qP in the leaf was observed at PPFD higher than 200 µmol m −2 s −1 with values reaching zero at 1585 µmol m −2 s −1 .The pitcher had lower qP values compared to the leaves of 0.88 to 0.42 at PPFD of 50-200 µmol m −2 s −1 .The qP of zero was observed at about PPFD of 900 µmol m −2 s −1 .The ETR values of the leaves increased sharply to a maximum of 60 µmol electrons m −2 s −1 at a PPFD of 500 µmol m −2 s −1 after which the ETR values decreased gradually (Figure2(b)).Similarly, the ETR of the pitcher increased rapidly to a maximum of 22 µmol electrons m −2 s −1 at a PPFD of 200 µmol m −2 s −1 after which it decreased gradually.These results show that the pitchers have a lower light utilisation compared to that of the leaves.

1 )Figure 1 :
Figure 1: Photosynthetic light response curve of leaf and pitcher of N. alata under different PPFDs.Means of 4 measurements from 4 different leaves and 4 different pitchers.Vertical bars represent standard errors.When the standard error bars cannot be seen, they are smaller than the symbols.
) and 4(b)).It is also interesting to see that TSP concentrations of both leaves and pitchers showed the same patterns of Chl content after different N treatments for 4 weeks (Figures4(c) and 4(d)).

Figure 2 :
Figure 2: Changes in qP and qN (a) and ETR (b) of leaf and pitcher of N. alata under different PPFDs.Means of 4 measurements from 4 different leaves and 4 different pitchers.Vertical bars represent standard errors.When the standard error bars cannot be seen, they are smaller than the symbols.

Figure 3 :
Figure 3: Changes in P max (a), (b) and Chl content (c), (d) of leaves (a), (c) and pitchers (b), (d) of N. alata after 4 weeks of different N treatments.Means of four measurements were obtained from four different leaves from four different plants.Means with different letters above the columns are statistically different (P < 0.05) as determined by Tukey's multiple comparison test.

Figure 4 :
Figure 4: Changes in TRN (a), (b) and TSP (c), (d) concentration of leaves (a), (c) and pitchers (b), (d) of N. alata following 4 weeks of different N treatments.Means of 4 measurements were obtained from 4 different leaves from 4 different plants.Means with different letters above the columns are statistically different (P < 0.05) as determined by Tukey's multiple comparison test.

Table 1 :
Total Chl content, Chl a/b ratio, total carotenoid content, and Chl/carotenoid ratio of leaf and pitcher of N. alata.The means and standard errors of four readings are given for each pigment.Letters represent comparison between leaf and pitcher.Any two means having a common letter are not significantly different.