Biochemical Systematics and Ecology 37 (2009) 707–715

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Biochemical Systematics and Ecology

journal homepage: www.elsevier .com/locate/biochemsyseco

Organ-specific and environmental control of accumulation ofpsychollatine, a major indole alkaloid glucoside fromPsychotria umbellata

Juçara T. Paranhos a,1, Variluska Fragoso a, Vivian C. da Silveira a, Amelia T. Henriques b,Arthur G. Fett-Neto a,*

a Laboratorio de Fisiologia Vegetal, Centro de Biotecnologia e Departamento de Botanica, Universidade Federal do Rio Grande do Sul,Av. Bento Gonçalves 9500, 91509-900, Caixa Postal 15005, Porto Alegre, RS, Brazilb Faculdade de Farmacia, Universidade Federal do Rio Grande do Sul, Av. Ipiranga 2752, 90610-000, Porto Alegre-RS, Brazil

a r t i c l e i n f o

Article history:Received 23 April 2009Accepted 5 December 2009

Keywords:Psychotria umbellataStress responseOrgan distributionMonoterpene indole alkaloidPsychollatine

* Corresponding author. Tel.: þ55 51 3308 7642;E-mail address: fettneto@cbiot.ufrgs.br (A.G. Fet

1 Present address: Centro de Ciencias Naturais eSanta Maria, RS 97105-900, Brazil.

0305-1978/$ – see front matter � 2009 Elsevier Ltddoi:10.1016/j.bse.2009.12.003

a b s t r a c t

Psychollatine is an unusual indole alkaloid produced by Psychotria umbellata Vell, anAtlantic Forest understorey tree. Leaves, young inflorescences and fruit pulp displayed highamounts of psychollatine (from 2.5 to 4.5% dry wt). Seeds accumulated lower quantities(0.2% dry wt), and roots, trace amounts (0.06% dry wt). Alkaloid accumulation was notseasonally affected and leaf content was not significantly enhanced by wounding, exposureto salicylic acid, hydrogen peroxide, or UV. The alkaloid content in leaves decreased uponcutting exposure to auxin. Under strong UV, the amount of chlorophylls did not decreasecompared to control contents at 48 and 72 h. Post-harvest psychollatine content in leaveswas stable in different temperatures, except at 65 �C. Psychollatine was an effective singletoxygen, superoxide, and peroxide quencher. These antioxidant properties and theconstitutively high amount of psychollatine indicate a role for the alkaloid in oxidativestress responses.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Alkaloids may be important in the interactions of plants with other organisms, such as microbes, insects and other plantspecies (Facchini, 2001). Alkaloid biosynthesis in plants is often tightly controlled during development and in response toenvironmental stresses, providing a way to regulate energy and nutrient investment in secondary metabolism (Pasquali et al.,2006). There is some overlapping between the signaling pathways of biotic and abiotic stresses in plants, often converging atthe level of reactive oxygen species (ROS) production (Shinozaki and Dennis, 2003).

Jasmonic acid (JA) and salicylic acid (SA) are two plant stress signal molecules that operate as endogenous messengersimplicated in plant resistance responses (Zhao et al., 2005). JA is produced following cell damage or herbivory via theoctadecanoid pathway and it can act as an efficient inducer of indole alkaloid accumulation in Psychotria brachyceras(Gregianini et al., 2004). SA and methyl salicylate also have important signaling roles in plant defense against pathogens,leading to systemic acquired resistance (SAR) (Heil and Ton, 2008) and stimulation of secondary metabolite yield, such asterpenes in pine oleoresin (Rodrigues and Fett-Neto, 2009).

fax: þ55 51 3308 7309.t-Neto).Exatas, Departamento de Biologia, Universidade Federal de Santa Maria,

. All rights reserved.

J.T. Paranhos et al. / Biochemical Systematics and Ecology 37 (2009) 707–715708

Tryptophan decarboxylase (TDC), a key enzyme in biosynthesis of the precursor tryptamine needed for monoterpeneindole alkaloid production, can be inhibited by auxin at the level of transcription (Facchini, 2001). Besides its role in indolealkaloid biosynthesis, TDC has also been implicated in one of several possible pathways of auxin biosynthesis (Bartel, 1997).

The Atlantic Forest, which extends from the northern to southern coast of Brazil, is one of the most endangered biodi-versity hot spot biomes of the world (Morellato and Haddad, 2000). The mild climate and the altitudinal gradient oftenassociated with this forest provide the conditions for the development of a rich variety of species (Basso et al., 2005). As partof an effort to describe and identify useful molecules from this ecosystem, a chemical screening, combining ethnobotanicaland chemotaxonomical leads, was carried out with species of the genus Psychotria, trees that grow in the understorey(Pasquali et al., 2006).

Psychollatine (Fig. 1) represents a new subclass of monoterpene indole alkaloids with a geniposide-derived terpenemoiety, and its occurrence has been reported for Psychotria and Palicourea species (Kerber et al., 2008; Narine and Maxwell,2009). It is an unusual monoterpenoid indole alkaloid, isolated from leaves of Psychotria umbellata Vell. This alkaloid showeddose dependent opioid-like analgesic activity, ansiolytic, antipsychotic and antimutagenic properties (Pasquali et al., 2006;Fragoso et al., 2008).

In order to gain insights on the metabolism, accumulation dynamics, and potential functions of this new type of alkaloid,the production profile in planta and the induced changes associated with stress treatments, as well as the distribution profileof psychollatine in vegetative and reproductive organs of P. umbellata, were investigated. Potential regulatory factors ofalkaloid biosynthesis were examined, such as mechanical wounding, exposure to UV, application of hydrogen peroxide,salicylic acid or the auxin 2,4-dichlorophenoxy acetic acid. The in vitro antioxidant activities of psychollatine against singletoxygen, superoxide and peroxide were also analyzed.

2. Material and methods

2.1. Plant materials

P. umbellata Vell. adult plants, growing in the understorey of the southern limit of the Atlantic Forest (Torres-RS, Brazil,29�20007" S and 49�43037" W) were used in the experiments. A voucher specimen is deposited in the UFRGS herbarium (ICN98869). For the seasonal study, nine permanently marked individuals had expanded leaves sampled at each season of theyear, for two consecutive years (2001 and 2002). Each plant was considered an experimental block and three replications oftwo leaves each were harvested per block.

For the determination of psychollatine distribution and concentration, leaves, stems, roots and immature inflorescencessamples were harvested in early summer (December). In early fall (March), new samples of leaves, stems and roots wereharvested together with green fruit samples. The mature fruits and seeds were collected in late fall (May). Immediately afterharvest, samples were placed in liquid nitrogen (N2) and frozen at �5�C until alkaloid analysis. With the exception of leaves,plant materials were lyophilized overnight prior to alkaloid extraction.

To investigate the post-harvest stability of psychollatine, leaves were stored for seven days in the dark under the followingconditions: freezing in liquid nitrogen and transfer to freezer (�5 �C); direct transfer to freezer without liquid nitrogenexposure; direct transfer to oven (65 �C) and maintenance at room temperature (25–30 �C). In this last condition, leaf samplesalso remained for six weeks and psychollatine extraction and analysis was carried out right after harvest and at every week.

2.2. Clonal propagation and regulation of alkaloid production

Tip cuttings from field-grown plants were harvested on consecutive years for clonal propagation by hydroponic rooting.Cuttings were rooted by transient exposure to 4-3-indolyl butyric acid (IBA) in 0.1�Murashige and Skoog (MS) salts (Kerberet al., 2001). The effect of salicylic acid (0.5 and 1.0 mM) and hydrogen peroxide (H2O2) (0.25, 0.5 and 1.0 mM) was inves-tigated separately by adding aqueous solutions of these compounds to the nutrient medium and by spraying plants with the

Fig. 1. Structure of the glucosidic monoterpene indole alkaloid psychollatine from Psychotria umbellata.

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same solutions to the point of imminent run off. These procedures were applied once at the start of the experiment (mid-morning). Control treatments were kept in nutrient medium only and were sprayed with water.

To cause wounding, about 75% of the total leaf area of rooted cuttings was damaged with tweezers and scissors. The effectof 2,4-dichlorophenoxy acetic acid (2,4-D) exposure on alkaloid accumulation was tested with a group of cuttings sponta-neously rooted without IBA pulse. After rooting, cuttings were exposed to 2,4-D (10 mg L�1). In experiments with salicylicacid, mechanic damage and 2,4-D, leaves were harvested every 48 h for psychollatine analysis, whereas in the study withhydrogen peroxide, this was done every 24 h. For high energy UV experiments, cuttings were supplemented with UV-Cradiation (16 h per day, 10.5 kJ cm�2), provided by a germicidal lamp, whereas control plants were maintained under whitelight only. Fully expanded leaves were harvested every 24 h for analysis of psychollatine content. Leaf discs (1.77 cm2) wereharvested for analysis of chlorophylls a and b (Gregianini et al., 2003). For all of the experiments, cuttings were maintainedin a growth room at 25� 2�C, irradiance (photosynthetically active radiation) of w73 mmol m�2 s�1 provided by whitefluorescent tubes and photoperiod of 16 h per day.

2.3. Psychollatine extraction and analysis

Tissue samples (approximately 1.0–2.0 g fw or 0.5–1.0 g lyophilized wt) were ground with mortar and pestle in liquidnitrogen. Tissues were extracted in 3 ml methanol (HPLC grade), mixed and sonicated for 30 min. The extracts werecentrifuged at 5000� g for 10 min and the supernatant was recovered. Chemical analysis of the methanolic extracts wasperformed using a Perkin–Elmer S200 HPLC equipped with UV detector (280 nm). Alternatively, a Waters Alliance 2690 HPLCsystem with photodiode array detector (PDA) was used. The analyses were carried out using a C18 reverse phase column(ODS Hypersyl), and the mobile phase consisted of solvent A (MeOH–H2O–trifluoroacetic acid (TFA), 20:80:0.05) and solventB (MeOH–TFA, 100:0.05) in a 20 min linear gradient from 100% A to 100% B; flow rates were 0.2 and 0.4 ml min�1, respec-tively for Waters Alliance 2690 HPLC system and Perkin–Elmer S200 HPLC equipment. For alkaloid quantification, 10 mLsamples of each extract were injected and an external standard curve was generated with pure psychollatine isolated fromleaves (Both et al., 2002). Peak identity was based on retention time, UV spectra, and co-chromatography with authenticpsychollatine.

2.4. Quenching of singlet oxygen assay, superoxide and peroxide tests

The quenching of singlet oxygen assay determined the ability of psychollatine to inhibit the self-sensitized photo-oxidation of rubrene by visible light, essentially as previously described (Gregianini et al., 2003). The O2 (1Dg) quenchingactivity has been determined for psychollatine using chloroform and the results compared with the corresponding values forDABCO (1,4-diazabicyclo[2.2.2]octane), which is a well-known O2 (1Dg) quencher. The concentrations used were 8� 10�4 M ofrubrene (Acros Organics, New Jersey, USA), 1 mM of DABCO (Acros Organics, New Jersey, USA) and 0.5 mM of authenticpsychollatine; chloroform (Nuclear, Sao Paulo, Brazil) was used as solvent. All solutions were prepared fresh for each assay. Totest for the involvement of chemical quenching, authentic psychollatine in methanol was irradiated with UV light for 2 and15 min, and analyzed by HPLC for evidence of degradation.

A peroxide test was conducted to examine the antioxidative properties of psychollatine. Peroxide activity was determinedby a reaction between ferrous ammonium sulfate aqueous solution and potassium thiocyanate at 0.1 M, both in the psy-chollatine plus rubrene solution and in the pure rubrene solution after 7 min of UV exposure. Controls of both solutions werenot exposed to UV. The peroxide oxidizes ferrous ion to ferric ion, which reacts with thiocyanate ion to give the characteristicred color of the complex (Morrison and Boyd, 1992).

peroxideþ Feþ2/Feþ3/ FeðSCNÞnð3—nÞ—Red

The ferrous iron oxidation was favored by shaking, since the peroxide was present in the non polar phase. The supernatant(polar phase) was measured in spectrophotometer at 660 nm.

For the analysis of psychollatine antioxidative activity against superoxide, 0.5 mM of the alkaloid was tested according toBeyer and Fridovich (1987) using Trolox 0.5 mM as a positive control. The production of purple formazan was determined bythe increase in absorbance at 560 nm after 10 min illumination from fluorescent lamps. The percentage of reductionprotection of the samples was based on the complete NBT reduction observed in the solvent control. The initial OD of eachsample was subtracted from their respective values after 10 min of light treatment.

2.5. Statistical analysis

The experiments on psychollatine distribution, seasonal patterns and post-harvesting treatments were performed withfive replicates and the studies on regulation of biosynthesis had four replicates. For the quenching, superoxide and peroxidetests, measures were done in duplicates. All experiments were independently repeated 2–3 times with similar results. Datawere transformed to log10 to obtain variance homogeneity when required and subjected to simple or factorial analysis ofvariance (ANOVA) and Duncan Test at P� 0.05 (when appropriate), using the statistical software SPSS 10.0.

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3. Results

3.1. Content of psychollatine, organ distribution and impact of reproductive phase-shift

Psychollatine concentration and distribution in adult field-grown plants was found to differ in the various plant partsevaluated. In vegetative tissues (Fig. 2A), leaves displayed the highest amounts of psychollatine (3.67� 0.5% dry wt,mean� standard deviation), followed by stems (1.01�0.5% dry wt). Roots had the lowest alkaloid amounts (0.06� 0.03% drywt), suggesting that its biosynthesis is predominant in shoots or that it undergoes rapid transport to aerial parts.

In reproductive tissues (Fig. 2B) psychollatine accumulation was shown to depend on the developmental stage. Immatureinflorescences yielded the highest alkaloid content (4.52� 0.5% dry wt) in the plants. At this same time of sampling, leaves,stems and roots accumulated lower amounts of psychollatine than in the vegetative period, 2.52� 0.2, 0.30� 0.1 and0.05� 0.07% dry wt respectively. At the beginning of fruit setting, the alkaloid content was lower than in later stages. Thegreen infructescences accumulated 1.07� 0.5% dry wt, whereas, at fruit maturation, the pulp had a psychollatine content of2.85� 0.05% dry wt. The lowest concentrations were observed in seeds (0.21�0.02% dry wt).

3.2. Seasonal accumulation profile of alkaloid and plant to plant variation

The analysis of alkaloid content, independent of the harvested plant, showed that there were no significant differencesthroughout the seasons in two consecutive years examined (data not shown). On the other hand, independent of the har-vesting season, field-grown plants showed statistical differences in accumulation of this alkaloid. For example, plant numbersix accumulated the highest mean psychollatine content (3.53� 0.38% dry wt), whereas plant number four had the lowestquantity (2.65� 0.41% dry wt).

3.3. Post-harvest stability of alkaloid in leaves

The post-harvest experiment showed that psychollatine in detached leaves is very stable (Fig. 3). The only temperaturethat significantly affected psychollatine content in leaves was 65 �C, resulting in a 2.5-fold decrease in the content (0.96% drywt), probably due to alkaloid degradation, although there were no significant changes in the UV spectra of the psychollatinepeak (data not shown).

3.4. Response of alkaloid content to stress factors and signaling molecules

Mechanic damage did not significantly change psychollatine content in leaves. Psychollatine contents did not show a clearoverall pattern of response to exposure to salicylic acid at 0.5 mM and 1.0 mM. Similarly, the hydrogen peroxide concen-trations tested did not significantly affect psychollatine content.

Fig. 2. Distribution of psychollatine in different plant parts of P. umbellata. A. Vegetative tissues: leaf, stem and root. B. Reproductive tissues: immature inflo-rescence, green infructescence, fruit pulp and seed. Bars within each graph sharing a letter are not different by a Duncan test (P� 0.05).

Fig. 3. Psychollatine accumulation in leaves of P. umbellata after different post-harvesting procedures. Bars sharing a letter are not different by a Duncan test(P� 0.05).

J.T. Paranhos et al. / Biochemical Systematics and Ecology 37 (2009) 707–715 711

In the experiments with 2,4-D, there was no significant interaction between exposure time and 2,4-D in the accumulationof psychollatine. Auxin exposure significantly decreased psychollatine concentrations at various exposure times (Fig. 4).

The accumulation of psychollatine in leaves of P. umbellata was not significantly affected by UV radiation (Fig. 5A). In threeexperiments, there was no significant difference between psychollatine accumulation in control or UV exposed leaves duringthe sampling period. However, in two experiments, a trend towards decreased alkaloid content was observed with exposuretime.

In spite of the long exposure to a very damaging quantity of UV-C, pigment concentrations were not significantly reduced.The contents of chlorophylls a and b increased initially (24 h) after the start of exposure, followed by a decrease at 72 h(Fig. 5B), maintaining pigment concentrations equivalent to those of the control treatment.

3.5. Antioxidant properties of psychollatine

If the absorbance decrease rate for rubrene in the presence of a putative quencher compound is smaller than in the control,this indicates that the compound is capable of capturing singlet oxygen, and protecting rubrene against photo-oxidation.Psychollatine clearly reduced the absorbance decrease rate of rubrene compared to rubrene alone (Fig. 6A), although DABCO(the positive control) was even more effective. The measures of singlet oxygen quenching activity for DABCO and psy-chollatine after one minute exposure were 1.21�0.36�108 and 1.74� 0.33�107, respectively (means� standard error of themeans); therefore, DABCO quenching activity was 6.96 times higher than that of psychollatine.

The rubrene assay was also carried out under UV-C light. It was observed that the absorbance decrease rate of rubrene withpsychollatine was slower than that when DABCO was present in the rubrene solution (data not shown). Moreover, it wasconsistently observed that the solution of psychollatine and rubrene after 2 min of UV exposure showed increased absor-bance when compared with untreated rubrene, exposed rubrene and exposed DABCO plus rubrene solution. At 4 min of UVexposure, control rubrene solution decreased 48% of the initial concentration whereas the solution of rubrene plus psy-chollatine decreased only 6%. Under these conditions, singlet oxygen quenching activity of psychollatine was 61 times higherthan that of DABCO (1.98� 0.25�109 and 3.25� 0.72�107, respectively). HPLC analyses of psychollatine methanolic solutionexposed to singlet oxygen (generated by UV irradiation) after a few minutes indicated significant degradation, suggesting theoccurrence of at least some chemical quenching by the alkaloid.

In order to test whether psychollatine could act as a peroxide-reducing agent, as suggested by the initial rise in rubreneconcentration when incubated with the alkaloid under UV, a test for the detection of peroxides was carried out. It was

Fig. 4. Psychollatine accumulation in leaves of rooted cuttings exposed to 2,4-D (10 mg L�1). Dark bars: untreated control; white bars: 2,4-D-treated. Zeroexposure time: content at harvest. Bars sharing a letter are not different by a Duncan test (P� 0.05).

Fig. 5. Psychollatine (% dry wt) and total chlorophyll contents (mg cm�2) in leaves of rooted cuttings of Psychotria umbellata exposed to UV-C radiation (16 h perday). Dark bars: untreated control (without UV); white bars: UV-treated. Zero exposure time: content at harvest. Bars within each graph sharing a letter are notdifferent by a Duncan test (P� 0.05).

J.T. Paranhos et al. / Biochemical Systematics and Ecology 37 (2009) 707–715712

observed that after 2 min of UV exposure, the solution of psychollatine plus rubrene showed 2.2 fold less peroxide than thesolution with pure rubrene. In the control, without UV radiation, the peroxide amount was 24 fold less in the presence ofpsychollatine.

Psychollatine was also able to mitigate the oxidative radical superoxide (Fig. 6B). Although less effective than the Troloxpositive control, psychollatine afforded approximately 50% scavenging of superoxide.

Fig. 6. Quenching properties of psychollatine. A. Typical kinetics of rubrene absorbance decrease due to singlet oxygen activity upon white light exposure incontrol (rubrene only, empty circles) and quenched solutions (DABCO – empty squares and psychollatine – filled squares). B. NBT protection assay – superoxidequenching. Bars sharing a letter are not different by a Duncan test (P� 0.05).

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4. Discussion

In agreement with the low psychollatine content in roots of field-grown plants, the absence of psychollatine in light anddark-grown rhizogenic calli (Paranhos et al., 2005) suggests that roots are not the biosynthetic site of this alkaloid. Psy-chollatine distribution profile was in good agreement with that of vindoline and catharanthine in Catharanthus pusillus(Zarate et al., 2001). In C. pusillus, leaves are the major repository site of these alkaloids, whereas stems and roots were lessimportant as accumulation organs.

The alkaloid-distribution results suggest that, at the start of the reproductive phase, plants are more active in accumu-lating psychollatine, or that there is transport of the alkaloid from vegetative to reproductive tissues. In addition, a dynamicaccumulation profile was apparent, with the high alkaloid content observed in immature inflorescences falling after flowerfertilization (transition between inflorescence to infructescence stage), followed by a new increase in alkaloid content inmature fruit. High accumulation of alkaloid in inflorescences was also reported for brachycerine in P. brachyceras (Gregianiniet al., 2004). This pattern may provide plants with better capacity to endure environmental stresses, improving reproductivesuccess. Alkaloids may also participate in mitigating oxidative stress generated by various biotic and abiotic stimuli (Pasqualiet al., 2006).

Psychollatine amounts in the various parts, particularly leaves, can be considered high when compared with the majormonoterpene indole alkaloid contents in other species of the same genus. Leaves of P. umbellata yielded psychollatine ataverage amounts 10 and 1.5 fold higher than the quantity of brachycerine in P. brachyceras and N,b-D-glucopyranosil vin-cosamide in Psychotria leiocarpa, respectively (Gregianini et al., 2004; Henriques et al., 2004). Compared to contents ofvinblastine and vincristine in C. roseus (approximately 0.5% dry wt), amounts of psychollatine are considerably higher (Zarateet al., 2001).

The lack of seasonal variation in psychollatine accumulation in field-grown plants contrasts with what was observed forbrachycerine in P. brachyceras, which had highest leaf contents in the fall of the first year of analyses (Gregianini et al.,2004). The lack of significant seasonal effects on psychollatine accumulation in P. umbellata suggests a phytoanticipin typeof accumulation profile. On the other hand, the detection of individuals in the plant population that consistently hadstatistically different overall contents of leaf psychollatine is in agreement with what was reported for leaf brachycerinecontents in P. brachyceras, highlighting the genetic component in the control of alkaloid accumulation (Gregianini et al.,2004).

Negative effects of high temperatures on post-harvest secondary product stability have been reported previously, perhapsdue to degradation by enhanced initial activation of non-specific enzymes, such as peroxidases and esterases (Fett-Neto andDiCosmo, 1992). The low temperature treatment (�5 �C) did not affect the levels of psychollatine (2.26–2.38% dry wt). Theincubation at 25–30 �C did not significantly reduce psychollatine content (2.06% dry wt), independently of the period ofstorage examined (data not shown). The chemical stability of the alkaloid in detached leaves kept for several days atcommonly observed temperatures in the field (25–30 �C) may support a putative contribution of the alkaloid to allelopathicaction, since the extract of P. umbellata leaves has been shown to be phytotoxic (Correa et al., 2008).

Although jasmonic acid (JA) and its octadecanoid precursors have been implicated as intermediate signals in many cases ofdamage-induced secondary metabolite accumulation (Gregianini et al., 2004; Zhao et al., 2005), damage did not increasepsychollatine content. Salicylic acid, which has been reported to stimulate secondary metabolism in C. roseus cell suspensioncultures (Godoy and Loyola, 1997), was not able to change alkaloid content in P. umbellata. Contrary to what was observed forpsychollatine concentration, H2O2 stimulated indole alkaloid accumulation in C. roseus cell cultures after 24 h of incubation,reaching highest contents after 48 h of treatment (Zhao et al., 2001).

The decrease in alkaloid concentration observed under auxin exposure is in agreement with reports on C. roseus cellcultures (Goddijn et al., 1992), which have related this response to down-regulation of tdc gene transcription. TDC ispresumably involved in the assembly of the indole moiety of psychollatine.

The lack of significant effects of UV-C exposure on psychollatine content is in contrast to the strong induction (10-foldincrease) observed for brachycerine in P. brachyceras (Gregianini et al., 2003). Increases in accumulation of other terpeneindole alkaloids upon UV treatment were also described by Ouwerkerk et al. (1999).

P. umbellata displayed considerable tolerance to the oxidative damage induced by UV-C exposure, since there was notsignificant decrease in chlorophyll content, in spite of some leaf loss. In P. brachyceras rooted cuttings, the same conditionshave been capable of causing severe damage and significant reduction (20%) in chlorophyll content (Gregianini et al., 2003).Several studies have shown that photosynthetic pigments may be adversely affected by UV radiation (Jansen et al., 1998).However, in the intertidal green alga Ulva expansa (Setch.), chlorophyll a and carotenoids increased upon UV-B exposure(Grobe and Murphy, 1998). A transient increase in chlorophyll content was also observed in P. umbellata.

It is possible that the high alkaloid content may explain the maintenance of chlorophyll levels and the lack of increase ofpsychollatine upon UV exposure, since basal concentrations of the alkaloid may be sufficient to effectively shield against someUV effects. Interestingly, UV-induced brachycerine accumulation brings its amounts in P. brachyceras to a range close to thebasal content of psychollatine in P. umbellata. Indole terpene alkaloids, such as brachycerine, are effective chromophores andmay act as UV filters or as free-radical scavengers (Gregianini et al., 2003; Nascimento et al., 2007). In fact, psychollatine hasbeen shown to quench hydroxil radical in vitro, and protect yeast mutants defective in antioxidant enzyme defenses againststress (Fragoso et al., 2008). In the present study, psychollatine was also shown to have broad antioxidant activity, beingeffective against peroxide, superoxide and singlet oxygen.

J.T. Paranhos et al. / Biochemical Systematics and Ecology 37 (2009) 707–715714

In spite of the differences in rate constants for singlet oxygen quenching between psychollatine and DABCO, the alkaloidhad significant activity against the ROS. Whereas there are secondary amines in psychollatine, DABCO contains tertiaryamines, which are more effective in quenching. In addition, the nitrogen of the indole portion of psychollatine formsa heterocycle, which is located in a simple indole without substitutions; this group usually reacts much more slowly with O2

(1Dg) (Monroe, 1977). Other potential quenching sites in psychollatine include the glucose residue and double bonds. Psy-chollatine showed higher quenching activity than the related alkaloid brachycerine from P. brachyceras (Gregianini et al.,2003), which has a very similar structure, but contains only one double bond in the terpene moiety. The experimentalevidence suggests that in planta psychollatine would have a similar reducing effect against oxidative stress.

In conclusion, the alkaloid predominated in aerial parts, particularly leaves, which constitute a source of the alkaloid. Thealkaloid was detected in lowest amounts in roots. The post-harvest experiments indicated that, unless exposed to fast dryingat high temperatures, psychollatine leaf content is rather stable. Psychollatine accumulation in leaves appears to beconstitutive and little regulated by stress factors, whereas the transition from vegetative to reproductive stage affects alkaloidaccumulation and distribution. Its presumed protective role could be related to ROS quenching capacity, shielding plant tissuefrom stress, such as photochemically induced oxidative damage, besides its UV absorbing properties, acting as a UV filter.

Acknowledgements

The authors thank the Brazilian Scientific Agencies CNPq, FAPERGS and CAPES for financial support to conduct thisresearch. These agencies were not involved in study design or development. We are grateful to Prof. Dr. L.R.M Baptista (Dept.of Botany, UFRGS) for allowing plant harvests in his property, to Dr. Marcos Sobral (Biology Institute, Universidade Federal deMinas Gerais, Brazil) for assistance in plant identification, and to Dr. Jose C. Fonseca Moreira (Graduate Program inBiochemistry, UFRGS) for the gift of a sample of Trolox.

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