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PlantaDOI 10.1007/s00425-014-2072-8

OrIgInal artIcle

Structural and functional characteristics of S‑like ribonucleases from carnivorous plants

Emi Nishimura · Shinya Jumyo · Naoki Arai · Kensuke Kanna · Marina Kume · Jun‑ichi Nishikawa · Jun‑ichi Tanase · Takashi Ohyama

received: 20 February 2014 / accepted: 24 March 2014 © Springer-Verlag Berlin Heidelberg 2014

recombinant carnivorous plant enzymes showed opti-mum activities at about pH 4.0. generally, poly(c) was digested less efficiently than poly(a), poly(I) and poly(U). the kinetic parameters of the recombinant D. muscipula enzyme (DM-I) and A. thaliana enzyme rnS1 were simi-lar. the kcat/Km of recombinant rnS1 was the highest among the enzymes, followed closely by that of recombi-nant DM-I. On the other hand, the kcat/Km of the recom-binant S. leucophylla enzyme was the lowest, and was ~1/30 of that for recombinant rnS1. the magnitudes of the kcat/Km values or kcat values for carnivorous plant S-like rnases seem to correlate negatively with the dependency on symbionts for prey digestion.

Keywords Droseraceae · enzyme kinetics · nepenthaceae · Phylogenetic tree · Protein structure · recombinant protein

AbbreviationsDtt DithiothreitoleDta ethylenediaminetetraacetic acidIPtg Isopropyl β-D-thiogalactopyranosidePcr Polymerase chain reactionrnase ribonuclease

Introduction

rnases of the t2 family are found in a variety of genomes, including plants, animals, protozoans, bacteria, fungi and viruses (Deshpande and Shankar 2002; luhtala and Parker 2010; MacIntosh et al. 2010). enzymes of this family are acidic endoribonucleases and are typified by rnase t2, an extracellular rnase from Aspergillus oryzae (Sato and egami 1957). t2 rnases have a wide spectrum of

Abstract although the S-like ribonucleases (rnases) share sequence homology with the S-rnases involved in the self-incompatibility mechanism in plants, they are not associated with this mechanism. they usually function in stress responses in non-carnivorous plants and in car-nivory in carnivorous plants. In this study, we clarified the structures of the S-like rnases of Aldrovanda vesiculosa, Nepenthes bicalcarata and Sarracenia leucophylla, and compared them with those of other plants. at ten positions, amino acid residues are conserved or almost conserved only for carnivorous plants (six in total). In contrast, two positions are specific to non-carnivorous plants. a phy-logenetic analysis revealed that the S-like rnases of the carnivorous plants form a group beyond the phylogenetic relationships of the plants. We also prepared and charac-terized recombinant S-like rnases of Dionaea muscipula, Cephalotus follicularis, A. vesiculosa, N. bicalcarata and S. leucophylla, and rnS1 of Arabidopsis thaliana. the

the nucleotide sequences reported in this paper have been submitted to DDBJ under accession numbers aB872467, aB872468 and aB872469.

Electronic supplementary material the online version of this article (doi:10.1007/s00425-014-2072-8) contains supplementary material, which is available to authorized users.

e. nishimura · S. Jumyo · n. arai · K. Kanna · M. Kume · t. Ohyama Major in Integrative Bioscience and Biomedical engineering, graduate School of Science and engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, tokyo 162-8480, Japan

J. nishikawa · J. tanase · t. Ohyama (*) Department of Biology, Faculty of education and Integrated arts and Sciences, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, tokyo 162-8480, Japane-mail: ohyama@waseda.jp

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biological functions, including scavenging nucleotides and phosphate for growth and metabolism; responding to phos-phate limiting conditions, senescence, or pathogen attack; serving in the self-incompatibility mechanism of plants; and modulating host immune responses (Deshpande and Shankar 2002; luhtala and Parker 2010; MacIntosh et al. 2010).

S-rnases and S-like rnases are rnase t2 fam-ily members. S-like rnases have sequence homology to S-rnases, but they differ in function. S-rnases function in self-incompatibility, which is a genetically controlled mechanism that prevents self-fertilization and promotes outcrossing and is found in Solanaceae, rosaceae and Scrophulariaceae (Mccubbin and Kao 2000). S-like rnases are not involved in the self-incompatibility mechanism. expression of S-like rnases is usually induced by phos-phate starvation, wounding or senescence in non-carnivo-rous plants (löffler et al. 1993; taylor et al. 1993; Bariola et al. 1994; Köck et al. 1995; Dodds et al. 1996; lers et al. 1998; Bodenhausen and reymond 2007; MacIntosh et al. 2010). However, we recently found that these enzymes are used for carnivory in carnivorous plants.

australian carnivorous plants Drosera adelae (sun-dew), which traps and digests insects with a sticky diges-tive liquid, and C. follicularis, which uses a pitcher filled with digestive enzymes for trapping and digesting prey, secrete S-like rnases Da-I and cF-I, respectively, in their digestive liquids, even in an ordinary state (Okabe et al. 2005a, b; nishimura et al. 2013). D. muscipula (Venus flytrap) also contains the S-like rnase DM-I in its digestive liquid (nishimura et al. 2013). the genes da-I encoding Da-I and cf-I encoding cF-I are highly active in each trap/digestion organ and inactive in other organs. their ortholog dm-I of D. muscipula becomes active only in its trap/digestion organ after trapping of prey. the regulatory mechanisms of the da-I, dm-I and cf-I expressions may differ and are not correlated with the phylogenetic relationship (nishimura et al. 2013). the confined expression of da-I in the glandular tenta-cles (trap/digestion organ of D. adelae) can be explained in terms of methylation/non-methylation of the promoter. In contrast, a transcription factor specifically expressed in the trap/digestion organ has been hypothesized for each regulation of dm-I and cf-I expressions (presumably, the factor for cf-I expression is constitutively expressed and that for dm-I expression is expressed in response to trapping of prey) (nishimura et al. 2013). Furthermore, conformational analysis using DIaMOD (Dlakic and Harrington 1998) suggested that the dm-I and cf-I pro-moters have highly curved conformations (nishimura and Ohyama unpublished results). curved Dna struc-tures are often implicated in the mechanism of transcrip-tion activation (Sumida et al. 2006; Kamiya et al. 2007).

thus, the conformations of the dm-I and cf-I promoters may also be implicated in the activation mechanism of transcription.

although genetic research on S-like rnases of car-nivorous plants has made some advances, their structural characteristics and enzymatic properties have been poorly understood. therefore, the current study was performed with two major goals: to identify common structural fea-tures among these S-like rnases and to understand their enzymatic properties. We first examined the gene and pro-tein structures of S-like rnases of A. vesiculosa, N. bical‑carata and S. leucophylla, which allowed a comparative study of six carnivorous plants enzymes. For the second goal, we prepared recombinant S-like rnases of five car-nivorous plants including D. muscipula, C. follicularis and recombinant rnS1 of A. thaliana (Bariola et al. 1994) and performed comparative characterizations, including deter-mination of kinetic parameters. In the protein structures, we identified ten positions at which only carnivorous plants use common or almost common amino acids and two positions that are specific for non-carnivorous plants. the kcat/Km of recombinant rnS1 was the highest among the enzymes and the value of recombinant DM-I was close to it. On the other hand, the kcat/Km of recombinant S. leuco‑phylla S-like rnase was the lowest. the kinetics of the car-nivorous plant enzymes seems to be consistent with their methods of prey digestion.

Materials and methods

Plants

Dionaea muscipula ellis, Cephalotus follicularis labill., Nepenthes bicalcarata Hook. f. and Sarracenia leuco‑phylla raf. were purchased from Yamada carnivorous plant nursery (Hiroshima, Japan). Aldrovanda vesiculosa l. was purchased from Pastime nursery (Hiroshima, Japan). the growth conditions of D. muscipula and C. follicularis were reported previously (nishimura et al. 2013). N. bicalcarata was grown in pure Sphagnum moss. to keep high humid-ity, pots with these plants were placed in a terrarium, which were placed indoors. temperature in the terrarium was kept at 26–28 °c throughout the year. S. leucophylla was grown in a soil mix [peat moss:loamy soil, 4:1 (v/v)]. the pot was placed outdoor. A. vesiculosa was grown in a water-filled earthenware pot, which was placed outdoors. all plants were grown without fertilizer.

Determination of av-I, nb-I and sl-I nucleotide sequences

genomic Dna from A. vesiculosa, N. bicalcarata and S. leucophylla was prepared by a conventional method.

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at first, partial nucleotide sequences of av-I, nb-I and sl-I genes, which were amplified by Pcr using each genomic Dna and degenerate primers (Supplementary table S1), were determined. then, using inverse Pcr (iPcr)-mediated chromosome walking, the nucleotide sequences of av-I, nb-I and sl-I genes and their 5′ and 3′-flanking regions were determined. to digest genomic Dna for iPcr, HindIII (for A. vesiculosa) or BglII (for N. bicalcarata and S. leucophylla) was used. the primer sets used in the iPcr are shown in Supplementary table S2. DDBJ accession numbers: av-I, aB872467; nb-I, aB872469; sl-I, aB872468. Detailed information is available upon request.

analyses of amino acid sequence identities and phylogenetic relationships

the amino acid sequences of the S-like rnases were aligned using clustal W. Pairwise amino acid sequence identity was calculated using the protein–protein BlaSt (BlaStp) algorithm, from the national center for Bio-technology Information (ncBI). the phylogenetic tree was constructed using the maximum-likelihood method with the Mega software, version 5.0 (tamura et al. 2011), with 1,000 bootstrap replicates.

construction of S-like rnase expression plasmids

Constructs for recombinant DM‑I, CF‑I, NB‑I and SL‑I expression

Using total rna prepared from each plant and primer sets shown in Supplementary table S3, dm-I, cf-I, nb-I and sl-I cDnas lacking the region encoding a putative signal peptide were prepared. each resulting Pcr fragment was digested with NdeI and BamHI, gel purified, and cloned between the NdeI and BamHI sites of the pet15b plas-mid (novagen), for the expression of an n-terminally His-tagged proteins.

Constructs for recombinant AV‑I and RNS1 expression

genomic A. thaliana Dna was purchased from Zyagen, and genomic A. vesiculosa Dna was prepared by a con-ventional method. Using each genomic Dna, the exons 2, 3 and 4 were amplified by Pcr with primers shown in Supplementary table S3. they were ligated in order. the resulting fragment was then ligated to an exon 1 fragment lacking the signal peptide-encoding sequence, which was prepared by annealing oligonucleotides shown in Supplementary table S3. the resulting fragment was

b

ed

ca

Fig. 1 carnivorous plants used in the study. a Aldrovanda vesiculosa, b Nepenthes bicalcarata, c Sarracenia leucophylla, d Dionaea muscipula, and e Cephalotus follicularis

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cloned between the NdeI and BamHI sites of the pet15b plasmid.

all plasmid structures were verified by sequencing. Fur-ther details of construct generation are available upon request.

expression and purification of recombinant proteins

each S-like rnase expression plasmid was transformed into E. coli strain Bl21(De3) (novagen). cells were grown in lB media containing 50 µg/ml ampicillin at 37 °c. expression of recombinant proteins was induced by addi-tion of 1 µM IPtg (isopropyl β-D-thiogalactopyranoside) at an A600 = 0.4–0.6. after incubation for 4 h, cells were harvested and recombinant proteins were purified using a MagneHis Protein Purification System (Promega). Purified proteins were buffer-exchanged into a solution containing 10 mM sodium citrate buffer (pH 5.0), 1 mM Dtt and 50 % glycerol. their molar concentrations were determined by the values of A280 and the following extinction coeffi-cients: DM-I, 44,515 M−1 cm−1; cF-I, 40,045 M−1 cm−1; aV-I, 44,515 M−1 cm−1; nB-I, 46,005 M−1 cm−1; Sl-I, 43,025 M−1 cm−1; rnS1, 47,035 M−1 cm−1, which were determined by using the ProtParam tool of the exPaSy server (http://web.expasy.org) (gasteiger et al. 2005).

Determination of rnase activity

rnase activity was assayed according to the method of Okabe et al. (2005a) and corbishley et al. (1984), with slight modifications. Briefly, using 2 μl of each protein solution described above, the assay was performed in 80 mM sodium citrate buffer (pH 4.0) with 16 μg puri-fied Torula yeast rna (Sigma–aldrich) in a final volume of 100 μl. after 4 min incubation at 37 °c, 100 µl of 5 % perchloric acid/lanthanum aqueous solution [lanthanum nitrate, 0.65 % (w/v) in perchloric acid] was added to stop the reaction and precipitate rna. Stopped reactions were held on ice for more than 20 min, and insoluble rna was removed by centrifugation for 10 min at 10,000g. the amount of solubilized rna was analyzed by measurement of A260 of the supernatant fraction, with the t = 0 control used as the A260 = 0.00 standard (blank). One ribonucle-ase unit was defined as the amount of enzyme causing an increase in ΔA260 of 1.0 min−1 cm−1 ml−1 at 37 °c, accord-ing to Wilson (1975).

Determination of pH and temperature optima

to determine optimum pH, each reaction was performed at 37 °c for 10 min in a volume of 250 μl containing 40.2 µg Torula yeast rna (Sigma–aldrich), 0.1–0.3 U of recom-binant enzyme (DM-I, aV-I, Sl-I and rnS1, 0.1 U; cF-I, 0.2 U; nB-I, 0.3 U) and 80 mM sodium citrate (pH 3.0–6.0) or citrate–phosphate buffer (pH 2.5). to determine opti-mum temperature, each reaction was performed at a given temperature ranging between 20 and 90 °c using 0.1 U of recombinant enzyme and 80 mM sodium citrate buffer at pH 4.0. the other conditions were the same as those used in the determination of optimum pH. In the both experiments, each reaction mixture was pre-incubated for 5 min at a given pH or temperature before adding a recombinant enzyme and the reaction was started with adding the enzyme and mixing the solution. the reaction was stopped by addition of 250 μl of perchloric acid/lanthanum solution, and the amount of solubilized rna was analyzed as described above.

Digestion of homopolyribonucleotides

Polyadenylic acid [poly(a)], polyinosinic acid [poly(I)], polycytidylic acid [poly(c)] and polyuridylic acid [poly(U)] were used as substrates. each reaction mixture (15 μl) contained 7.5 µg of substrate, 6.0 × 10−2 U of enzyme and 50 mM sodium acetate buffer (pH 4.0). the reactions were performed at 25 °c for 5 min, 30 min or 1 h, and the reaction products were electrophoresed on a 1 % agarose gel in 1/2 × tBe (45 mM tris–borate/1 mM eDta, pH 8.0). rna molecules in the gel were stained with methylene blue.

Fig. 2 comparison of deduced amino acid sequences of aV-I, nB-I and Sl-I with those of other S-like rnases. amino acid sequences of 19 class I S-like rnases are aligned. they are as follows: DM-I of D. muscipula (nishimura et al. 2013), Da-I of D. adelae (Okabe et al. 2005a), cF-I of C. follicularis (nishimura et al. 2013), ZrnaseII and ZrnaseI of Z. elegans (Ye and Droste 1996), PD2 and PD1 of P. dul‑cis (Ma and Oliveira 2000; Van nerum et al. 2000), ne of N. alata (Dodds et al. 1996), nW and ngr3 of N. glutinosa (Hayashi et al. 2003), le and lX of L. esculentum (löffler et al. 1993; Köck et al. 2004), rnS1, rnS3 and rnS5 of A. thaliana (Bariola et al. 1994; theologis et al. 2000) and PtrrnS1 of P. trichocarpa (tuskan et al. 2006). Colons indicate amino acid residues identical to those of DM-I. Dashes indicate gaps introduced to maximize the sequence iden-tity. c1–c5 (green boxes) indicate regions that are highly conserved among S-like rnases (Kao and Huang 1994; Shimizu et al. 2001). the two histidine residues important for rnase activity are shaded in yellow. cysteine residues involved in disulfide bond formation are marked with red asterisks (tanaka et al. 2000). Putative secretion sig-nal sequences are underlined. these assignments were made accord-ing to the cited reports or predicted by the bioinformatics tools in the SignalP 4.0 program (Petersen et al. 2011). the putative second-ary structures deduced by the Phyre software (Kelley and Sternberg 2009) are shown above the sequences. the conserved amino acid residues (≥18/19 matches) are shaded in gray. amino acid residues that seem specific or near-specific for carnivorous plants are shaded in magenta with the following criteria: specific, the six carnivorous plants use an identical amino acid, while the non-carnivorous plants tend to use another amino acid; near-specific, five carnivorous plants except S. leucophylla use an identical amino acid that is not used in most of the non-carnivorous plants. amino acid residues that are conserved only among the non-carnivorous plants are shaded in light blue. nB-I has four amino acid residues at positions 183–185 indi-cated as XXX and six residues at 219–223 indicated as ZZZZZ, which are ntnY and tngVtD, respectively

▸

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assay for sensitivity to eDta

reactions were performed at 25 °c for 30 min in a vol-ume of 15 μl containing 7.5 µg poly(a), 6.0 × 10−2 U of enzyme and 50 mM sodium acetate buffer (pH 5.0), in the presence of 10 or 25 mM eDta or in the absence of eDta. rna molecules were stained with methylene blue.

Kinetic assay

Kinetic assays were carried out with varying concentrations (0.45–3.6 μM) of baker’s yeast trna (Sigma–aldrich) in 80 mM sodium citrate buffer (pH 4.0) and 0.17–3.60 pmol of each recombinant enzyme (DM-I, 0.17 pmol; aV-I, 1.12 pmol; nB-I, 0.42 pmol; cF-I, 1.43 pmol; Sl-I, 3.60 pmol; rnS1, 0.24 pmol) in a final volume of 120 μl at 37 °c. the substrate was pre-incubated in a reaction buffer at 37 °c for 10 min, and the reaction was started with add-ing the enzyme solution and mixing the reaction mixture. at given time points, the reactions were stopped by addi-tion of 120 μl of perchloric acid/lanthanum solution and the amount of solubilized rna was analyzed as described above. Values for Km and Vmax were determined from lineweaver–Burk plots, and kcat values were calculated with the following formula: kcat = Vmax/[E], where [E] is the enzyme concentration used.

Results

gene and protein structures of A. vesiculosa, N. bicalcarata, S. leucophylla S-like rnases

Photographs of A. vesiculosa, N. bicalcarata, S. leuco‑phylla, D. muscipula and C. follicularis are shown in Fig. 1. We first identified the S-like rnase genes of A. vesiculosa, N. bicalcarata and S. leucophylla and named them as av-I, nb-I and sl-I, respectively. these genes have four exons and three introns (Supplementary Fig. S1), simi-larly to da-I, dm-I and cf-I (Okabe et al. 2005b; nishimura et al. 2013). they are categorized as class c genes (Ma and Oliveira 2000) or class I genes (Igic and Kohn 2001) and regarded as the most evolved S-like rnase genes (Ma and Oliveira 2000).

the amino acid sequences of the enzymes aV-I, nB-I and Sl-I encoded by av-I, nb-I and sl-I were deduced based on the cDna sequences. aV-I, nB-I and Sl-I con-tain 228, 231 and 226 amino acids and have molecular masses of 25.1, 25.5 and 25.1 kDa, pI values of 4.29, 4.13 and 4.52, and signal peptides of 22, 25 and 23 amino acids, respectively. the amino acid sequences of aV-I, nB-I and Sl-I were aligned with those of Da-I, DM-I and cF-I and with other class I S-like rnases

(those from monocots were excluded), such as Zrna-seII and ZrnaseI (Zinnia elegans: a common zinnia) (Ye and Droste 1996), rnases PD2 and PD1 (Prunus dul‑cis: almond) (Ma and Oliveira 2000; Van nerum et al. 2000), rnase ne (Nicotiana alata) (Dodds et al. 1996), rnase nW and ngr3 (Nicotiana glutinosa) (Hayashi et al. 2003), rnase le and lX (Lycopersicon escu‑lentum: tomato) (löffler et al. 1993; Köck et al. 2004), rnase rnS1, rnS3 and rnS5 (A. thaliana) (Bariola et al. 1994; theologis et al. 2000) and PtrnS1 (Popu‑lus trichocarpa: black cottonwood) (tuskan et al. 2006) (Fig. 2). aV-I, nB-I and Sl-I also have structural charac-teristics of S-like rnases, including conserved histidine residues (shaded in yellow in Fig. 2), five highly con-served regions (c1–c5) (Kao and Huang 1994; Shimizu et al. 2001), and five pairs of cysteine residues that form disulfide bonds (marked with red asterisks in Fig. 2): cys45-cys51, cys52-cys108, cys81-cys127, cys190-cys226, cys206-cys217 (tanaka et al. 2000).

the current study also identified many conserved amino acid residues (18/19 or 19/19 matches; shaded in gray in Fig. 2), irrespective of the differences between carnivorous plants and non-carnivorous plants. Several of them form regions that are located between α-helices, β-strands or these two structures or are within these struc-tures (Fig. 3). In our previous report (nishimura et al. 2013), we reported 19 positions that seemed specific or highly specific for carnivorous plants’ S-like rnases. as shown in Fig. 2, ten positions still stand among them, in spite of the increased number of samples (eight S-like rnases were added in total). namely, at each of four positions, 85, 111, 115 and 135, the six carnivorous plants use an identical amino acid, while the non-carnivorous plants tend to use another amino acid (shaded in magenta in Fig. 2). at each of six positions, 48, 105, 164, 171, 172 and 179 (also shaded in magenta in Fig. 2), five car-nivorous plants, except for S. leucophylla, use an identi-cal amino acid that is not used in most (at 48, 171, 172, 92.3 %; at 179, 76.9 %; at 105, 164, 69.2 %) of the non-carnivorous plants. Positions 93 and 186 (shaded in light blue in Fig. 2) seemed to be specific for the non-carnivo-rous plants: they use the same amino acids at these posi-tions (93, Ser; 186, Pro), but the carnivorous plants do not show this conservation. the locations of these residues in the 3D structure are shown in Fig. 3.

aV-I, nB-I and Sl-I were found to share 59–84 % identities to Da-I, DM-I and cF-I (Fig. 4a), which very roughly correlates with the phylogenetic relationships among the six plants (ellison and gotelli 2009), and 52–70 % identities to the other class I S-like rnases, as shown in Supplementary table S4. the phyloge-netic relationships of the S-like rnases estimated by the maximum-likelihood method (“Materials and methods”)

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revealed that the S-like rnases DM-I, aV-I, Da-I, nB-I, cF-I and Sl-I of the carnivorous plants form a group (Fig. 4b). although P. trichocarpa and C. follicularis are phylogenetically close (Fig. 4a), their enzymes, PtrrnS1 and cF-I, respectively, are not similar to each other (Fig. 4b), and the latter resembles the S-like rnases of the other carnivorous plants. therefore, the phylogenetic relationships among the plants do not seem to be greatly implicated in the similarities in their enzyme structures, which is further discussed later.

Optimum pH and optimum temperature

the characteristics of the native enzyme Da-I of D. adelae have been described (Okabe et al. 2005a). thus, the cur-rent study was performed to characterize the enzymes of the other carnivorous plants shown in Fig. 1, using recom-binant enzymes. Optimum activities occurred at about pH 4.0 for D. muscipula DM-I, A. vesiculosa aV-I and C. folli‑cularis cF-I and about pH 3.5 for N. bicalcarata nB-I and S. leucophylla Sl-I (Supplementary Fig. S2). A. thaliana

Fig. 3 Predicted 3D structure of S-like rnases. the structure was modeled using the Phyre software (Kelley and Stern-berg 2009) and visualized by chimera (Pettersen et al. 2004). the coloring is the same as that used in Fig. 2. Briefly, green: five conserved regions of S-like rnases; yellow: two histidine residues important for rnase activity; gray: the conserved amino acid residues identified in the current study; magenta: amino acid residues that are specific or near-specific for carnivorous plants; light blue: amino acid residues conserved only among the non-carnivorous plants

S93

P186

S48

S105

N85

S111

S115

E135

N164

D172

S171Q179

C5

H66H124

Front view

Back view

H66

H124

N85

E135

S93

S105

S111 S115

S48

P186D172

S171

N164

C4

C5

C4

Q179

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rnS1 also had optimum activity at about pH 4.0, but retained ~50 % activity at pH 6.0, while the S-like rnases of carnivorous plants lost or almost lost their activities at pH 6.0. In contrast, these S-like rnases still showed some activity at pH 2.5, at which rnS1 lost its activity.

the optimum pH values of the recombinant DM-I, nB-I and cF-I enzymes are very close to the pH values of their digestive liquids of 3.9 (takahashi et al. 2009), 3.9–4.0 (measured in the current study) and ~4.0 (measured in the current study), respectively. S. leucophylla is not likely to secrete a digestive liquid (anderson and Midgley 2003; Peroutka et al. 2008; adlassnig et al. 2011) and we were unable to collect digestive liquid from A. vesiculosa. It was also of interest that similar pH vs. activity curves were obtained for DM-I and aV-I and also for nB-I, cF-I and Sl-I.

the effect of temperature on the enzymatic activity is shown in Supplementary Fig. S3. Under the experimental conditions used, most enzymes showed the highest activi-ties at ~50–60 °c. the temperature vs. activity profiles of aV-I and cF-I were similar to each other and slightly dif-ferent from the profiles of the other enzymes. However, there was no clear difference between the enzymes of carnivorous plants and rnS1. It is noteworthy that nB-I retained almost full activity at 70 °c and >80 % activity at 80 °c.

Substrate specificity and effect of eDta

the substrate specificity of the recombinant enzymes was examined using homopolyribonucleotides (Fig. 5). all enzymes digested poly(a), poly(U) and poly(I) much more easily than poly(c). therefore, poly(c) may be an unfavorable substrate for the S-like rnases examined. Many plant rnases are insensitive to eDta (Bariola and green 1997), and this is also the case for Da-I (Okabe et al. 2005a). thus, we examined whether the recombinant enzymes prepared in this study could digest poly(a) in the presence of eDta. all the enzymes were found to be insensitive to eDta (data not shown).

Kinetics

For S-like rnase kinetic studies, the initial rate of hydroly-sis (v) was obtained at five trna concentrations (range 0.45–3.6 μM) (Supplementary Fig. S4), and lineweaver–Burk plots of initial velocity vs. trna concentration were obtained for recombinant DM-I, aV-I, nB-I, cF-I, Sl-I and rnS1 to determine Km and kcat (insets in Supplemen-tary Fig. S4). as shown in table 1, the kinetic parameters of recombinant DM-I and rnS1 were similar. the speci-ficity constant (kcat/Km) of recombinant rnS1 was the highest among the enzymes, followed closely by the value

of recombinant DM-I. On the other hand, the kcat/Km of recombinant Sl-I was the lowest (~1/30 of that for recom-binant rnS1), followed closely by recombinant cF-I with the second lowest value. the kcat/Km values of recombinant nB-I and aV-I were between those of recombinant DM-I and cF-I. We could not detect any correlation between these values and the phylogenetic relationships among the plants.

Discussion

Structural characteristics of the S-like rnases from carnivorous plants

the S-like rnases were divided into two classes, based on their gene structures (Igic and Kohn 2001; rojas et al. 2013). the class I S-like rnase genes have up to three introns, while the class II genes have more introns. the current study aligned the amino acid sequences of 19 class I S-like rnases, including six enzymes from carnivorous plants (Fig. 2), and newly revealed the “additional” con-served regions or amino acid residues [shown in gray; posi-tions with 18 or 19 (all) identical amino acid residues are shaded]. roughly, these conserved amino acids and regions are either located between α-helices, β-strands or these two structures or within these structures (Fig. 3), indicating that they contribute to structural maintenance. although the amino acid sequences in the c4 and c5 regions may not appear to be conserved regions, their locations in the 3D protein structure suggest that they may be conserved: the former constitutes an α-helix and the latter forms a β-strand (Fig. 3).

With the larger number of samples used for the align-ment of amino acid sequences, the carnivorous plant-specific or near-specific amino acid positions, which we reported previously (nishimura et al. 2013), were refined. nine positions were eliminated and ten were retained (shaded in magenta in Fig. 2). at four positions, 85, 111, 115 and 135, the six carnivorous plants use an identical amino acid (asn, Ser, Ser and glu, respectively), but most

Fig. 4 Phylogenetic relationships among plants and S-like rnases. a Phylogeny of the plants discussed in the current study. Part of the angiosperm phylogeny (Stevens 2001) containing the relevant plants is shown. rnase names are given in parentheses. the numerals are % identities between the S-like rnases. b Maximum-likelihood phylog-eny of S-like rnases. the amino acid sequences were aligned using clustal W, and the phylogenetic tree was constructed using the max-imum-likelihood method with the program Mega 5.0 (tamura et al. 2011). Bootstrap values are shown in the nodes, and were calculated by 1,000 replicates. Vrn1 (Volvox carteri) (Shimizu et al. 2001) was used as an outgroup. the S-like rnases of carnivorous plants are shown in red

▸

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of the non-carnivorous plants use another amino acid. asn, Ser and glu are polar or hydrophilic amino acids. at posi-tions 85 and 135, the non-carnivorous plants tend to use different hydrophilic or polar amino acids. at positions 111 and 115, the same tendency is observed for the non-carniv-orous plants, but non-polar amino acids were also observed (111 had gly in six cases; 115 had ala in four cases). thus, the asn, Ser and glu residues at the indicated positions

in the six carnivorous plants seem to be conserved for a structural reason, rather than for maintaining or enhanc-ing the aqueous solubility of each enzyme. at six positions, 48, 105, 164, 171, 172 and 179, a similar contrast is seen between carnivorous plants, except S. leucophylla, and non-carnivorous plants. at each of these positions, S. leu‑cophylla tends to have the major amino acids that are used among the non-carnivorous plants. the Sl-I enzyme (or digestive liquid) is not likely to be secreted from S. leuco‑phylla (anderson and Midgley 2003; Peroutka et al. 2008; adlassnig et al. 2011), in contrast to the S-like rnases of the other five carnivorous plants. thus, this may be reflected in the amino acid usage at these six positions.

In our previous report (nishimura et al. 2013), we also indicated six positions with amino acid residues that are “nearly” common among the eight non-carnivorous plants (>6/8 matches), but not common among the three carniv-orous plants (D. adelae, D. muscipula and C. follicularis; no identical residues were allowed among the three, but

DM-I

1 2 3 4 5 6 7 8 9 10 11 12 1314 15 16

poly(A) poly(C) poly(U) poly(I)

time

CF-I

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

poly(A) poly(C) poly(U) poly(I)

time

NB-I

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

poly(A) poly(C) poly(U) poly(I)

time

SL-I

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

poly(A) poly(C) poly(U) poly(I)

time

RNS1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

poly(A) poly(C) poly(U) poly(I)

time

AV-I

1 2 3 4 5 6 7 8 9 10 11 1213 14 15 16

poly(A) poly(C) poly(U) poly(I)

time

Fig. 5 Substrate specificity of recombinant S-like rnases. Using 6.0 × 10−2 U of each enzyme, the indicated homopolyribonucleo-tides were digested at 25 °c for 5 min (lanes 2, 6, 10 and 14), 30 min

(lanes 3, 7, 11 and 15), or 1 h (lanes 4, 8, 12 and 16). lanes 1, 5, 9 and 13 are undigested controls. Digestion was performed in sodium acetate buffer (pH 4.0)

Table 1 Kinetic parameters of recombinant S-like rnases

rnase Km (µM) kcat (min−1) kcat/Km (min−1 µM−1)

DM-I 0.66 179.0 271.2

aV-I 1.19 35.2 29.6

nB-I 1.85 118.5 64.1

cF-I 1.67 27.7 16.6

Sl-I 3.31 30.8 9.3

rnS1 0.48 151.0 314.6

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the matches to the residues of non-carnivorous plants were allowed). In the current study, we tightened the criterion for amino acid conservation for the non-carnivorous plants (13 proteins in seven plants), and screened for the positions where only multiple carnivorous plants break the conser-vation. as a result, we found two positions, 93 and 186, that meet this criterion (shaded in light blue in Fig. 2). Ser and Pro are used at these positions in the non-carnivorous plants, respectively. among the carnivorous plants, only S. leucophylla and C. follicularis use the same amino acids at these positions. at position 93, the other carnivorous plants have gln, and at 186 they use ala or gly. therefore, the properties of the amino acid residues are almost the same between the non-carnivorous plants and the carnivorous plants (Ser and gln, polar; Pro, ala and gly, non-polar). In addition, we could not detect any positional characteristics in the 3D structure. thus, these differences may not have any special meaning.

the homology analysis described above suggested that the S-like rnases of carnivorous plants share struc-tural similarities beyond the phylogenetic relationships among the plants. Indeed, they formed a group in the phy-logenetic relationship for proteins (Fig. 4b). although P. trichocarpa and C. follicularis are phylogenetically close (Fig. 4a), the cF-I enzyme from the latter species is much closer to the S-like rnases of the other carnivorous plants than to P. trichocarpa PtrrnS1 (Fig. 4b). In addition, the enzyme homologues from a single plant species, such as P. dulcis PD1 and PD2, L. esculentum le and lX, A. thaliana rnS1, rnS3 and rnS5, N. glutinosa nW and ngr3, and Z. elegans ZrnaseI and ZrnaseII, also sug-gest that the evolutional relationships among the plants do not have a large influence on the structures of the class I S-like rnases. In each set, the enzymes are not structurally similar (Fig. 4b). Instead, the functional similarity seems to be more implicated in the protein structure. rnases PD2, rnS1, le, ne and lX are induced by phosphate starvation (Jost et al. 1991; löffler et al. 1993; Bariola et al. 1994; Dodds et al. 1996; Ma and Oliveira 2000), while ZrnaseII, rnS1, le and nW are induced by wounding in common (Ye and Droste 1996; lers et al. 1998; leBrasseur et al. 2002; Hayashi et al. 2003) (rnS1 and le are induced by either stimulus). With the exception of lX, which is induced by phosphate starvation or senescence, the other six enzymes form a group (Fig. 4b). thus, the enzymes with a wounding-responsive or phosphate-starvation-responsive nature generally seem to have similar structures. ZrnaseI, lX and ngr3 also form a group. ZrnaseI is used in autol-ysis during xylogenesis (Ye and Droste 1996), and ngr3 responds to virus infection (Hayashi et al. 2003), which are different from responses to phosphate starvation, wounding or carnivory. the functions of PtrrnS1, PD1, rnS3 and rnS5 are unknown (Bariola et al. 1994; theologis et al.

2000; tuskan et al. 2006). thus, the protein functions seem to be more related to their structures than the evolutional relationships between the plants, at least for the class I S-like rnases. the prestin, a motor protein of the mamma-lian outer hair cells shows a similar phenomenon (liu et al. 2010). therefore, it seems safe to conclude that the S-like rnases of carnivorous plants form a group with similar structures and distinctive functions.

enzymatic characteristics of S-like rnases from carnivorous plants

the optimum pH values of the recombinant enzymes were as follows: D. muscipula DM-I, A. vesiculosa aV-I and C. follicularis cF-I, pH 4.0; and N. bicalcarata nB-I and S. leucophylla Sl-I, pH 3.5 (Supplementary Fig. S2). the native Da-I of D. adelae showed optimum activity at pH 4.4 (Okabe et al. 2005a). thus, the S-like rnases of the six carnivorous plants have optimum activities between pH 3.5–4.5, indicating that they are acidophilic. these values are reasonable for DM-I, nB-I, cF-I and Da-I, because they are very close to the pH values of their digestive liq-uids. On the other hand, the S-like rnases of the non-car-nivorous plants generally seem to be less acidophilic. their optimum pH values are reported as follows: native nW of N. glutinosa, ~6.0 (Kariu et al. 1998; Hino et al. 2002); native le of L. esculentum, 5.5 (Jost et al. 1991); recombi-nant rnS3 of A. thaliana, 6.0 (Hillwig et al. 2011); native lX of L. esculentum, 5.9 (löffler et al. 1992); and recom-binant ngr3 of N. glutinosa, ~5.0 (Hino et al. 2002). thus, their optimum activities are between pH 5.0–6.0. In addition, regarding nW and rnS3, 60–90 % activities were observed even at pH ~7.0 (Kariu et al. 1998; Hillwig et al. 2011). Furthermore, based on the pH vs. activity pro-files, the current study also suggested that the recombinant rnS1 of A. thaliana is less acidophilic than the enzymes from carnivorous plants (Supplementary Fig. S2). thus, the S-like rnases of carnivorous plants seem to be more acido-philic than those of the non-carnivorous plants.

Similar pH vs. activity curves were obtained for DM-I and aV-I, which both have “active” (snap) traps and are phylogenetically very close (Supplementary Fig. S2); and also for nB-I, cF-I and Sl-I, which all have pitfall traps, but are not necessarily phylogenetically close. thus, the insect-trapping or -digesting mechanism may have some relevance to the similarity in the observed pH vs. activity curves. However, we cannot currently explain the similar-ity in these profiles. as for the optimum temperature and sensitivity to eDta, the enzymes from the carnivorous plants seem to share common characteristics, except for the tolerance of nB-I to high temperatures (Supplementary Fig. S3). In the current study, poly(c) was poorly digested by all the enzymes examined (Fig. 5). In the cases of the

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native Da-I from D. adelae and rnase nW from N. gluti‑nosa, poly(c) digestion was not detected (Kariu et al. 1998; Okabe et al. 2005a). thus, poly(c) may be an unfavorable substrate for the class I S-like rnases.

the kinetics of the carnivorous plant enzymes seems to be consistent with their methods of prey digestion. Sarra-ceniaceae largely rely on symbionts for prey digestion, and their enzyme production is dubious or improbable (ander-son and Midgley 2003; Peroutka et al. 2008; adlassnig et al. 2011). thus, Sl-I is presumably not used for carnivory. this hypothesis can explain the lowest kcat/Km and the high-est Km values of the recombinant Sl-I (indicating lower cat-alytic efficiency and affinity to rna) among the enzymes examined. Similarly, Nepenthes and Cephalotus, which produce their own digestive enzymes, have symbionts that considerably contribute to prey digestion (adlassnig et al. 2011). the similar Km values of recombinant nB-I and cF-I may be explained in terms of the common features of their traps and prey digestion mechanisms employing symbionts. A. vesiculosa is a free-floating aquatic carnivorous plant. In this sense, it is similar to Utricularia purpurea, which hosts various symbionts (richards 2001). A. vesiculosa may also have symbionts, and if so, they may contribute to the diges-tion of trapped prey. among the carnivorous plants used in this study, presumably only D. muscipula digests prey without assistance from symbionts. the magnitudes of the kcat/Km values or the kcat values seem to correlate negatively with the dependency on symbionts for prey digestion.

Acknowledgments this work was supported by a research grant from the Ministry of education, culture, Sports, Science and tech-nology of Japan (MeXt) to t.O.

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