Isolation and Characterization of Cyclotides from Brazilian Psychotria: Significance in Plant Defense and Co-occurrence with Antioxidant Alkaloids Hélio N. Matsuura,† Aaron G. Poth,‡ Anna C. A. Yendo,† Arthur G. Fett-Neto,† and David J. Craik‡,* †Center for Biotechnology and Department of Botany, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil ‡Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia. 1 ABSTRACT Plants from the genus Psychotria include species bearing cyclotides and/or alkaloids. The elucidation of factors affecting the metabolism of these molecules as well as their activities may help to understand their ecological function. In the present study, high concentrations of antioxidant indole alkaloids were found to co-occur with cyclotides in Psychotria leiocarpa and P. brachyceras. The concentrations of the major cyclotides and alkaloids in P. leiocarpa and P. brachyceras were monitored following herbivore- and pathogen-associated challenges, revealing a constitutive, phytoanticipin-like accumulation pattern. Psyleio A, the most abundant cyclotide found in the leaves of P. leiocarpa, and also found in P. brachyceras leaves, exhibited insecticidal activity against Helicoverpa armigera larvae. Addition of ethanol in the vehicle for peptide solubilization in larval feeding trials proved deleterious to insecticidal activity, and resulted in increased rates of larval survival in treatments containing indole alkaloids. This suggests that plant alkaloids ingested by larvae might contribute to herbivore oxidative stress detoxification, corroborating, in a heterologous system with artificial oxidative stress stimulation, the antioxidant efficiency of Psychotria alkaloids previously observed in planta. Overall, the present study reports data for eight novel cyclotides, the identification of P. leiocarpa as a cyclotide-bearing species, and the absence of these peptides in P. umbellata. 2 Rubiaceae is among the plant families able to produce both cyclotides and alkaloids, with important representatives of each class of compounds being found in Oldenlandia affinis1 and Coffea arabica,2 respectively. Psychotria is the largest genus within the Rubiaceae family, distributed throughout tropical and subtropical regions of the world.3-5 Understanding the distribution, role, and possible synergies between indole alkaloids and cyclotides in Rubiaceae plants may be a useful tool to clarify their complex phylogeny and biological targets. Cyclotides are the most abundant naturally-occurring circular proteins in plants.6-7 To date, the main ecological role ascribed to cyclotides relates to their insecticidal properties,8-9 which are thought to arise by cyclotide-induced rupture of midgut epithelium cells.10 Their interaction with membrane lipids appears to occur in a receptor-independent manner,11 a feature that may prevent insects from acquiring resistance.12-13 Cyclotides contain 28 to 39 amino acids (www.cybase.org.au), and are characterized by their distinct structure: three disulfide bonds form a “cystine knot”14 embedded within the circular peptide backbone, which is made up of six loops between the cysteine residues (Figure 1). The structure is stabilized by hydrogen-bonding interactions with a conserved glutamic acid in loop 1.15 This structure engenders cyclotides with exceptional stability and has led to them being proposed as potential scaffolds in the design of target-specific drugs.12, 16 Biological effects described for naturally occurring cyclotides include uterotonic, anti-HIV, antimicrobial, insecticidal, and molluscicidal activities.10, 17-20 Figure 1. Cyclopsychotride A from Psychotria vellosiana (formerly P. longipes), the first cyclotide described in the genus Psychotria. 3 The described bioactivities of Psychotria cyclotides include: cytotoxic activity against MCF-7 and MCF-7/ADR breast cancer cell lines for psyle A, C and E (P. leptothyrsa);21 antimicrobial properties and hemolytic activity against human red blood cells for cyclopsychotride A (P. vellosiana);17 and inhibitory activity against prolyl-oligopeptidase by psysol 2 (P. solitudinum).22 To date, 16 cyclotide sequences have been identified across eight Psychotria species; namely, P. vellosiana (cyclopsychotride A),15 P. suterella (PS-1),6 P. leptothyrsa (psyles A-F),23 P. brachiata (psybra 1), P. deflexa (psydef 1 and 2), P. poeppigiana (psypoe 1), P. suerensis (psysue 1 and 2), and P. solitudinum (psysol 1 and 2).22, 24 In addition, six cyclotides (caripe 1–6) have been described from Carapichea ipecacuanha (formerly P. ipecacuanha).24 Other cyclotide-containing species, with sequences yet to be characterized, are: P. brachyceras, P. buchtienii, P. chiriquiensis, P. elata, P. goldmanii, P. mortoniana, P. pilosa, P. prunifolia, P. punctata and P. trichophora.6, 24 Sixty-one Psychotria species apparently lack cyclotides.6, 23-24 Psychotria species are also rich sources of alkaloids.25 Although alkaloids are often ascribed antiherbivore roles, the primary functions of these metabolites in some plants may in fact be in oxidative stress detoxification and control.26 As defense molecules, alkaloids can be neurotoxic or disrupt cell signaling.27-28 Shoots of the southern Brazilian Psychotria accumulate significant concentrations of indole alkaloids (0.2% to 4.5% dry weight) with strong antioxidant activity, but these metabolites are not herbivore deterrents.29-33 It is possible that in these plants a combined defense strategy is in play, with antioxidant alkaloids, both inducible34-35 and constitutive, acting in plant oxidative stress detoxification29-30 and cyclotides fulfilling the role of herbivore inhibition. The current study focused on the isolation, identification, and structural characterization of new cyclotides from selected Psychotria species, as well as on understanding their potential role in plant defense against insects. In addition, it was sought to better understand whether cyclotide and indole monoterpene alkaloid expression is integrated in the plant defense framework of two of the most common Psychotria species in the understory of the southern Atlantic Forest,36 P. leiocarpa and P. brachyceras. 4 RESULTS AND DISCUSSION Novel Cyclotides and a Novel Cyclotide-Producing Plant. Until now, cyclotides have not been found in pantropical Psychotria species, with the only exception being P. punctata, which is thought to be a cyclotide-containing species but for which no sequence has yet been reported.24 Of the four species analyzed in this study (P. brachyceras, P. leiocarpa, P. carthagenensis and P. umbellata), only P. brachyceras and P. leiocarpa were found to be cyclotide-producing plants (Table 1). No evidence for cyclotides in P. umbellata leaves was found, indicating it to be a cyclotide non-producing species, along with P. carthagenensis, which has previously been described as a cyclotide non- producing species.21 In P. leiocarpa, evidence for at least eight cyclotides was found and five complete sequences were obtained through sequencing based on MS/MS data from MALDI-TOF/TOF and static nanospray experiments, all of which were novel (psyleio A-E). Evidence for at least 17 cyclotides was obtained for P. brachyceras (Table 1). In total, seven complete sequences – three novel and unique to P. brachyceras, three shared with P. leiocarpa, and one known sequence (cycloviolacin O17 from Viola odorata) – were obtained. Evidence for all Psychotria cyclotide sequences is presented in the Supporting Information (Figures S1–S6). The new cyclotides were named following the nomenclature scheme proposed by Broussalis et al.,37 using the first letters of the genus and the specific epithet of each species. Cyclotides were alphabetically ordered based on yields observed in plant material. 5 Table 1. Cyclotides in Psychotria Species no. peptide native massa RA massa digested massa (Da) (Da) (Da) Psychotria. leiocarpa 1 psyleio A 2988.06 3336.16 3354.15 2 psyleio B 2948.06 3296.16 3314.15 3 psyleio C 2921.04 3269.16 3287.12 4 psyleio D 2925.02 3273.12 3291.11 5 psyleio E 3026.03 3374.15 3392.14 6 3088.97 3436.09 7 3266.13 3614.24 3632.23 8 3289.10 3637.24 Psychotria. brachyceras 1 psyleio A 2988.85 3336.94 3354.91 2 psyleio B 2948.85 3296.92 3314.92 3 psyleio D 2925.80 3273.89 3291.87 4 psybry A 3289.03 3637.10 3655.07 5 psybry B 3271.01 3619.66 6 3187.01 3535.09 7 2940.82 3288.90 8 cyO17 3153.04 3501.12 9 3227.99 3575.03 3593.98 10 psybry C 3231.99 3579.08 11 3281.14 3629.18 12 3291.02 3639.09 3657.09 13 3299.02 3647.12 14 3303.03 3651.10 15 3307.03 3655.10 16 3320.99 3668.07 17 3345.04 3693.13 a All presented masses correspond to uncharged masses (M0). Psychotria carthagenensis from southern Brazil lacks cyclotides, as reported for specimens from several locations,6, 23 and is also devoid of alkaloids.38 However, some variations in metabolite profiles have been described for plants originating from other regions. Specimens found in northern South America were used as a replacement for P. viridis as a source of the psychoactive alkaloid N,N- dimethyltryptamine. Cyclotide Yields in Plant Leaves. Based on yields of the isolation protocol, P. leiocarpa contained four major cyclotides: psyleio A–D. Psyleio A was present at a concentration of approximately 0.1% dry weight, and was at least twice as abundant as psyleio B (approximately 0.05% dry weight). P. brachyceras expressed three abundant cyclotides: psybry A, psyleio A and psybry B, with psybry B being around ten times less abundant (0.01% dry weight) than psybry A (approximately 0.1% dry weight); psyleio A was found in concentrations near 0.05% dry weight in P. brachyceras leaves. Psyleio B and D were found in P. brachyceras leaves in much lower concentrations. Representative sequencing and characterization data for the first characterized cyclotide in this study, psyleio A, are shown in Figure 2. 6 Figure 2. Characterization of psyleio A from Psychotria. leiocarpa. (A) MALDI-MS of Psychotria. leiocarpa extract. The putative cyclotide with a mass of 2989.3 Da was reduced and alkylated, and trypsin- or endoproteinase GluC-digested prior to MS/MS analysis.