Species of some genera of tropical plants growing in acidic and depleted soils form traps (pitchers) capable of capturing and digesting insects and even small vertebrates; these traps have arisen due to leaf modification during the evolutionary adaptation of plants to nutrient deficiencies [1, 2]. Despite the fact that carnivorous plants have evolved independently in several lines of flowering plants, all of them have pitchers of a similar structure [3]. To attract prey, carnivorous plants use volatile aromatic compounds and sweet compounds of protein nature [3], and the presence of lids and several layers of high-molecular-weight waxes on the inner surface does not allow the prey to escape from the pitcher [4]. The captured prey is digested by the hydrolytic enzymes (proteases, esterases, lipases, phosphatases, chitinases, etc.) secreted in the digestive zone of the pitcher [2]. Comparative analysis of the pitcher contents in carnivorous plants of the genera Cephalotus, Nepenthes, and Sarracenia revealed a similar set of proteins [3].

Since winged insects are the main prey of carnivorous plants, chitinases play a special role in the digestive process. Chitinases (EC 3.2.1.14) are a class of hydrolytic enzymes that catalyze the degradation of chitin to oligosaccharides [5]. The original role of plant chitinases is to protect them from chitin-containing pathogens [6]. These enzymes are considered a key evolutionary factor that provided the appearance of at least nine independent lineages of carnivorous plants that can digest insects under nutritional deficiency conditions. In addition to the protective function, chitinases in carnivorous plants are involved in biodegradation of insect chitin [3, 6].

Plant chitinases are a variable group of proteins united by the presence of a catalytic glycoside hydrolase (GH) domain but greatly differed in the primary sequence, domain composition, and cellular localization [6]. Based on amino acid sequence homology, presence of specific motifs, and biochemical characteristics, five (more rarely, seven) classes of chitinases are distinguished [7]. Classes I, II, IV, VI, and VII are characterized by the presence of domain GH19, whereas enzymes of classes III and V contain domain GH18 [7, 8]. Chitinases of the GH18 family are present in a wide range of organisms, such as viruses, bacteria, animals, and higher plants, whereas GH19 chitinases are found only in higher plants and some bacteria [5]. In addition to the catalytic domain, chitinases of classes I and IV contain N-terminal chitin-binding domain (CBD1) and interdomain proline/glycine-rich linker peptide, which varies both in length and composition [5, 7].

Carnivorous plants of the genus Nepenthes are used as models for studying predation mechanisms in plants for more than 100 years [9]. Despite the fact that the biochemical and transcriptome composition of the pitcher content in different Nepenthes species is actively studied [2, 5, 9], transcripts of only one or two genes encoding chitinases of classes I [1], III [2], and IV [10] have been identified and described to date, whereas more than 20 chitinase genes were found in the genomes of non-carnivorous plants [6, 11].

The aim of this work was to identify new chitinase genes in Nepenthes sp. and determine their expression levels in leaves and pitchers at different stages of development based on transcriptome data.

The search for the chitinase-coding sequences was performed in the transcriptomes of mature leaves and pitchers at three developmental stages— primordial pitcher (approximately 1 cm in height), unopened young pitcher (3–4 cm), and open mature pitcher (7–11 cm), in three biological replicates. The leaf was taken due to the fact that the pitcher is a modification of this organ. Since leaves and pitchers of Nepenthes are coated with a well-developed wax layer, which hampers extraction, to isolate RNA from Nepenthes tissues we developed a technique with the use of CTAB buffer containing 2% (w/v) CTAB (cetyltrimethylammonium bromide), 0.1 M Tris-HCl (pH 9.5), 20 mM EDTA, 1.4 M NaCl, and 1% beta-mercaptoethanol. Briefly, 7 mL of hot (65°C) CTAB buffer was added to 1 g of plant tissue ground in liquid nitrogen, the mixture was stirred and incubated at 65°C for 3 min. Then, obtained suspension was mixed with an equal volume of chloroform : isoamyl alcohol (24 : 1) and centrifuged at 11000 g for 15 min at 4°C. The supernatant was mixed with 0.25 vol of 10 M LiCl, incubated for 3 hours at –20°C, and centrifuged at 11000 g for 15 min at 4°C. The pellet, which contained RNA, was dissolved in 20 μL of deionized water (prepared in Milli-Q system, Millipore, United States). RNA preps were purified from possible genomic DNA using the RNase-free DNase Set (Qiagen, United States).

Prepared RNA samples were sequenced in an Illumina MiSeq platform (Core Facility “Bioengineering,” the Research Center of Biotechnology, RAS). The assembly and mapping of reads as well as the identification of the protein-coding genes in contigs was performed as described previously [12]. Gene expression level was calculated as the number of reads mapped on gene sequences per million reads, divided by the gene length. The statistical significance of differences in gene expression levels in different samples (leaf and three stages of pitcher development) was determined using the edgeR software. To identify primary chitinase sequences and determine the conserved domains and motifs, the NCBI database (http://blast.ncbi.nlm.nih.gov/) and MEME 4.11.2 software (http://meme-suite.org/tools/meme) were applied. N-terminal signal peptides were identified using the SignalP 4.1 software (http://www.-cbs.dtu.dk/services/SignalP). Comparative analysis of the amino acid sequences and the dendrogram construction were performed using the MEGA7.0 software (www.megasoftware.net).

In total, by the results of automatic annotation of 12 analyzed transcriptomes (leaf and three stages of pitcher development, three replicates), we identified 27 sequences homologous to plant chitinase genes. However only 15 of them contained the full-length coding part, i.e., had the start codon ATG and one of the stop codons (Table 1). The obtained sequences were deposited at the NCBI database (accessions MH430914–MH430928).

Table 1. Transcripts of chitinase genes and their expression profile in leaves and pitchers of Nepenthes sp.
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Based on the results of structural and phylogenetic analysis, the identified chitinase sequences were divided into two groups (Fig. 1). In chitinases NChi1, NChi2, NChi9, NChi10, and NChi11, chitin-binding (CBD1) and glycoside hydrolase (GH19) domains were identified. The remaining chitinases possessed only one domain (GH18 or GH19) was found (Fig. 1, Table 1). In addition, the analyzed chitinases (except for NChi5, NChi7, and NChi15) contained N-terminal signal peptides, which could be involved in the export of the enzyme from the cells to the fluid in the pitcher digestive zone. The comparative alignment with the known plant chitinase sequences from the NCBI database showed that Nepenthes sp. contains chitinases of all five known classes (Table 1). Previously, several class I, class III, and class IV chitinases have been identified and described in Nepenthes [1, 2, 10]. Comparative analysis revealed a high homology of known chitinases with the chitinases NChi1, NChi2, NChi9, NChi10, and NChi11, which were identified in the present study. The remaining ten chitinases (NChi3, NChi4, NChi5, NChi6, NChi7, NChi8, NChi12, NChi13, NChi14, and NChi15) were found in Nepenthes for the first time.

Fig. 1.
figure 1

Phylogenetic classification and structural analysis of the amino acid sequences of Nepenthes sp. chitinases. The dendrogram was generated by the maximum likelihood method (model WAG + G + I) using MEGA 7.0 software. Rectangles indicate the conserved motifs identified using MEME 4.11.2. Motifs 1–10 are localized in the GH19 domain, and motifs 11–16 are localized in the GH18 domain. * Chitin-binding domain.

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The amino acid sequences of chitinases NChi10 and NChi11 contained the chitin-binding domain (38 aa) and the GH19 domain (232 aa); in the latter, the functional motifs MLXXR, XFYTYX, AFXXAA, FXXTGX, and TSH, specific to chitinases GH19 of class I [13, 14], were found. In NChi1 and NChi9, the chitin-binding domain (27 aa) and GH19 domain (203 aa) were shorter than in the other chitinases. In the GH19 domain, motifs DXXXSFKXALW and GFGXTIRAIN were revealed, which allowed us to classify these chitinases into class IV [13]. Chitinases NChi3, NChi4, NChi5, NChi6, and NChi7 significantly varied in length (Table 1) and contained only the GH19 domain; based on this, they were classified into class II [13]. The analysis of nucleotide alignments showed that the chitinases encoded by NChi3, NChi5, NChi6, and NChi7 genes may be isoforms of NChi4, which apparently emerged as a result of alternative splicing.

The other chitinase family included NChi2, NChi8, NChi12, NChi13, NChi14, and NChi15, which contained the GH18 domain. Comparative analysis of their amino acid sequences with the known chitinases in the NCBI database classified chitinases NChi2, NChi12, NChi13, and NChi14 into class III, because their domain contained the active site LDGIDFDIE, characteristic of this class [15], and NChi15 was classified into class V.

MEME analysis of the conserved motifs in the sequences of Nepenthes sp. chitinases showed that GH18 group had a more conserved primary structure as compared to GH19 (Fig. 1). Interestingly, chitinase NChi8 contained the GH18 domain (according to NCBI-CDD) but had no motifs specific to any class of GH18 chitinases (according to [15]). On the dendrogram (Fig. 1), NChi8 formed the basal branch to the GH19 cluster. Possibly, NChi8 represents a new class of chitinases, which emerged as a result of evolutionary duplication and diversification of genes encoding GH18 chitinases in carnivorous plants.

Transcriptome analysis showed that the identified chitinase-coding genes differed by the expression level both between different organs and developmental stages and between each other. The maximum transcription level was found for the NChi4 gene (class II) in open mature pitchers (Table 1). The expression level of NChi4 and NChi3, NChi5, NChi6, and NChi7 genes, encoding NChi4 isoforms, increased during the pitcher development, reaching maximum in open mature pitchers (Table 1). Also, a high expression level was detected for the NChi11 gene (class IV); however, unlike NChi4, it was the highest in primordial pitchers and decreased as they developed (Table 1). Differences in the NChi4 and NChi11 expression levels between leaves and pitchers as well as between primordial pitchers and mature pitchers were statistically significant (p <0.05).

The other chitinase genes analyzed either had a low transcriptional activity or were not expressed at all three stages of pitcher development (Table 1).

Previous studies of Nepenthes chitinases were performed solely on pitchers [1, 2, 10]. In this study, transcriptome analysis showed that chitinase genes of all five classes are also expressed in the leaves, but at a lower level than in the pitchers. Apparently, a certain basic level of chitinase gene expression in leaves is required for protecting plants against pathogens, since chitinases primarily play a protective role in plants [6]. Note that in some replicates of mature leaf, an elevated expression (more than 40 units) of a number of identified genes, e.g., NChi1, NChi2, and NChi4, was detected (Table 1).

The NChi2-homologous class III chitinase, described previously in one of Nepenthes species, had a low enzymatic activity in the digestive fluid [2]. At the same time, invariability of its expression level in response to the substrate addition indicates the absence of the effect of this chitinase on insect digestion [2]. This was confirmed by our data, because the NChi2 transcription remained at about the same low level both in the pitcher (at all stages) and in the leaf, except for one replicate, in which the level increased more than 10 times (Table 1). This might be associated with the plant response to a local environmental factor.

Thus, in the transcriptomes of pitchers (at three stages of development) and mature leaves of Nepenthes sp., mRNA sequences of 15 chitinases belonging to classes I–V were identified and characterized. Chitinases of classes II and V in members of the genus Nepenthes were identified for the first time.

ACKNOWLEDGMENTS

This work was partially supported by the Russian Science Foundation (project no. 14-24-00175) and was performed using the experimental climate control facility located in the Institute of Bioengineering, Research Center of Biotechnology, Russian Academy of Sciences.

COMPLIANCE WITH ETHICAL STANDARDS

The authors declare that they have no conflict of interest. This article does not contain any studies involving animals or human participants performed by any of the authors.