Organ-specific expression and epigenetic traits of genes encoding digestive enzymes in the lance-leaf sundew (Drosera adelae).

Over the last two decades, extensive studies have been performed at the molecular level to understand the evolution of carnivorous plants. As fruits, the repertoire of protein components in the digestive fluids of several carnivorous plants have gradually become clear. However, the quantitative aspects of these proteins and the expression mechanisms of the genes that encode them are still poorly understood. In this study, using the Australian sundew Drosera adelae, we identified and quantified the digestive fluid proteins. We examined the expression and methylation status of the genes corresponding to major hydrolytic enzymes in various organs; these included thaumatin-like protein, S-like RNase, cysteine protease, class I chitinase, ß-1, 3-glucanase, and hevein-like protein. The genes encoding these proteins were exclusively expressed in the glandular tentacles. Furthermore, the promoters of the ß-1, 3-glucanase and cysteine protease genes were demethylated only in the glandular tentacles, similar to the previously reported case of the S-like RNase gene da-I. This phenomenon correlated with high expression of the DNA demethylase DEMETER in the glandular tentacles, strongly suggesting that it performs glandular tentacle-specific demethylation of the genes. The current study strengthens and generalizes the relevance of epigenetics to trap organ-specific gene expression in D. adelae. We also suggest similarities between the trap organs of carnivorous plants and the roots of non-carnivorous plants.

Biochemical and antifungal characteristics of recombinant class I chitinase from Drosera rotundifolia.

DrChit is class I chitinase involved in the digestion of insect prey of Drosera rotundifolia plants. Herein, we cloned the DrChit-S open reading frame lacking the 5'- sequence coding signal peptide into the pET32a vector and its derivate lacking the thioredoxin tag. After DrChit-S + Trx and DrChit-S-Trx overexpression in Escherichia coli cells and purification on Ni-NTA agarose, both enzymes exhibited maximum activity at pH 6.0 and 38 °C. Surprisingly, the DrChit -S + Trx exerted double enzyme activity and improved all kinetic parameters for FITC-chitin substrate degradation resulting in catalytic efficiency three times higher (46.2 mM-1. s-1) than DrChit-S-Trx (13.63 mM-1. s-1). The 3D-structure of DrChit-S + Trx revealed different spatial arrangement of the three tyrosine residues in chitin-binding site, while their aromatic rings showed better stacking geometry for CH/π interactions with the carbohydrate substrate. In contrast, there were no significant differences between both enzymes when the effect of metal ions and their antifungal potential were tested. Quantitative in vitro assays showed growth suppression of Fusarium poae (40%), Trichoderma viride (43.8%), and Alternaria solani (52.6%) but not Rhizoctonia solani (sp.). Our study indicates that sundew chitinase has potential in biotechnology either for degradation of chitin to oligomers applicable in medicine or for plant defense fortification.

Expression of Drosera rotundifolia Chitinase in Transgenic Tobacco Plants Enhanced Their Antifungal Potential.

In this study, a chitinase gene (DrChit) that plays a role in the carnivorous processes of Drosera rotundifolia L. was isolated from genomic DNA, linked to a double CaMV35S promoter and nos terminator in a pBinPlus plant binary vector, and used for Agrobacterium-mediated transformation of tobacco. RT-qPCR revealed that within 14 transgenic lines analysed in detail, 57% had DrChit transcript abundance comparable to or lower than level of a reference actin gene transcript. In contrast, the transgenic lines 9 and 14 exhibited 72 and 152 times higher expression level than actin. The protein extracts of these two lines exhibited five and eight times higher chitinolytic activity than non-transgenic controls when measured in a fluorimetric assay with FITC-chitin. Finally, the growth of Trichoderma viride was obviously suppressed when the pathogen was exposed to 100 µg of crude protein extract isolated from line 9 and line 14, with the area of mycelium growth reaching only 56.4% and 45.2%, of non-transgenic control, respectively. This is the first time a chitinase from a carnivorous plant with substrate specificity for long chitin polymers was tested in a transgenic plant with the aim of exploring its antifungal potential.

Protein structure networks provide insight into active site flexibility in esterase/lipases from the carnivorous plant Drosera capensis.

In plants, esterase/lipases perform transesterification reactions, playing an important role in the synthesis of useful molecules, such as those comprising the waxy coatings of leaf surfaces. Plant genomes and transcriptomes have provided a wealth of data about expression patterns and the circumstances under which these enzymes are upregulated, e.g. pathogen defense and response to drought; however, predicting their functional characteristics from genomic or transcriptome data is challenging due to weak sequence conservation among the diverse members of this group. Although functional sequence blocks mediating enzyme activity have been identified, progress to date has been hampered by the paucity of information on the structural relationships among these regions and how they affect substrate specificity. Here we present methodology for predicting overall protein flexibility and active site flexibility based on molecular modeling and analysis of protein structure networks (PSNs). We define two new types of specialized PSNs: sequence region networks (SRNs) and active site networks (ASNs), which provide parsimonious representations of molecular structure in reference to known features of interest. Our approach, intended as an aid to target selection for poorly characterized enzyme classes, is demonstrated for 26 previously uncharacterized esterase/lipases from the genome of the carnivorous plant Drosera capensis and validated using a case/control design. Analysis of the network relationships among functional blocks and among the chemical moieties making up the catalytic triad reveals potentially functionally significant differences that are not apparent from sequence analysis alone.

The role of electrical and jasmonate signalling in the recognition of captured prey in the carnivorous sundew plant Drosera capensis.

The carnivorous sundew plant (Drosera capensis) captures prey using sticky tentacles. We investigated the tentacle and trap reactions in response to the electrical and jasmonate signalling evoked by different stimuli to reveal how carnivorous sundews recognize digestible captured prey in their traps. We measured the electrical signals, phytohormone concentration, enzyme activities and Chla fluorescence in response to mechanical stimulation, wounding or insect feeding in local and systemic traps. Seven new proteins in the digestive fluid were identified using mass spectrometry. Mechanical stimuli and live prey induced a fast, localized tentacle-bending reaction and enzyme secretion at the place of application. By contrast, repeated wounding induced a nonlocalized convulsive tentacle movement and enzyme secretion in local but also in distant systemic traps. These differences can be explained in terms of the electrical signal propagation and jasmonate accumulation, which also had a significant impact on the photosynthesis in the traps. The electrical signals generated in response to wounding could partially mimic a mechanical stimulation of struggling prey and might trigger a false alarm, confirming that the botanical carnivory and plant defence mechanisms are related. To trigger the full enzyme activity, the traps must detect chemical stimuli from the captured prey.

Molecular characterization and evolution of carnivorous sundew (Drosera rotundifolia L.) class V ß-1,3-glucanase.

MAIN CONCLUSION: A gene for ß-1,3-glucanase was isolated from carnivorous sundew. It is active in leaves and roots, but not in digestive glands. Analyses in transgenic tobacco suggest its function in germination. Ancestral plant ß-1,3-glucanases (EC 3.2.1.39) played a role in cell division and cell wall remodelling, but divergent evolution has extended their roles in plant defense against stresses to decomposition of prey in carnivorous plants. As available gene sequences from carnivorous plants are rare, we isolated a glucanase gene from roundleaf sundew (Drosera rotundifolia L.) by a genome walking approach. Computational predictions recognized typical gene features and protein motifs described for other plant ß-1,3-glucanases. Phylogenetic reconstructions suggest strong support for evolutionary relatedness to class V ß-1,3-glucanases, including homologs that are active in the traps of related carnivorous species. The gene is expressed in sundew vegetative tissues but not in flowers and digestive glands, and encodes for a functional enzyme when expressed in transgenic tobacco. Detailed analyses of the supposed promoter both in silico and in transgenic tobacco suggest that this glucanase plays a role in development. Specific spatiotemporal activity was observed during transgenic seed germination. Later during growth, the sundew promoter was active in marginal and sub-marginal areas of apical true leaf meristems of young tobacco plants. These results suggest that the isolated glucanase gene is regulated endogenously, possibly by auxin. This is the first report on a nuclear gene study from sundew.

Structural and functional characterisation of a class I endochitinase of the carnivorous sundew (Drosera rotundifolia L.).

MAIN CONCLUSION: Chitinase gene from the carnivorous plant, Drosera rotundifolia , was cloned and functionally characterised. Plant chitinases are believed to play an important role in the developmental and physiological processes and in responses to biotic and abiotic stress. In addition, there is growing evidence that carnivorous plants can use them to digest insect prey. In this study, a full-length genomic clone consisting of the 1665-bp chitinase gene (gDrChit) and adjacent promoter region of the 698 bp in length were isolated from Drosera rotundifolia L. using degenerate PCR and a genome-walking approach. The corresponding coding sequence of chitinase gene (DrChit) was obtained following RNA isolation from the leaves of aseptically grown in vitro plants, cDNA synthesis with a gene-specific primer and PCR amplification. The open reading frame of cDNA clone consisted of 978 nucleotides and encoded 325 amino acid residues. Sequence analysis indicated that DrChit belongs to the class I group of plant chitinases. Phylogenetic analysis within the Caryophyllales class I chitinases demonstrated a significant evolutionary relatedness of DrChit with clade Ib, which contains the extracellular orthologues that play a role in carnivory. Comparative expression analysis revealed that the DrChit is expressed predominantly in tentacles and is up-regulated by treatment with inducers that mimick insect prey. Enzymatic activity of rDrChit protein expressed in Escherichia coli was confirmed and purified protein exhibited a long oligomer-specific endochitinase activity on glycol-chitin and FITC-chitin. The isolation and expression profile of a chitinase gene from D. rotundifolia has not been reported so far. The obtained results support the role of specific chitinases in digestive processes in carnivorous plant species.

Functional analyses of carnivorous plant-specific amino acid residues in S-like ribonucleases.

Unlike plants with no carnivory, carnivorous plants seem to use S-like ribonucleases (RNases) as an enzyme for carnivory. Carnivorous plant-specific conserved amino acid residues are present at four positions around the conserved active site (CAS). The roles of these conserved amino acid residues in the enzymatic function were explored in the current study by preparing five recombinant variants of DA-I, the S-like RNase of Drosera adelae. The kcat and kcat/Km values of the enzymes revealed that among the four variants with a single mutation, the serine to glycine mutation at position 111 most negatively influenced the enzymatic activity. The change in the bulkiness of the amino acid residue side-chain seemed to be the major cause of the above effect. Modeling of the three dimensional (3D) structures strongly suggested that the S to G mutation at 111 greatly altered the overall enzyme conformation. The conserved four amino acid residues are likely to function in keeping the two histidine residues, which are essential for the cleavage of RNA strands, and the CAS in the most functional enzymatic conformation.

S-like ribonuclease gene expression in carnivorous plants.

Functions of S-like ribonucleases (RNases) differ considerably from those of S-RNases that function in self-incompatibility. Expression of S-like RNases is usually induced by low nutrition, vermin damage or senescence. However, interestingly, an Australian carnivorous plant Drosera adelae (a sundew), which traps prey with a sticky digestive liquid, abundantly secretes an S-like RNase DA-I in the digestive liquid even in ordinary states. Here, using D. adelae, Dionaea muscipula (Venus flytrap) and Cephalotus follicularis (Australian pitcher plant), we show that carnivorous plants use S-like RNases for carnivory: the gene da-I encoding DA-I and its ortholog cf-I of C. follicularis are highly expressed and constitutively active in each trap/digestion organ, while the ortholog dm-I of D. muscipula becomes highly active after trapping insects. The da-I promoter is unmethylated only in its trap/digestion organ, glandular tentacles (which comprise a small percentage of the weight of the whole plant), but methylated in other organs, which explains the glandular tentacles-specific expression of the gene and indicates a very rare gene regulation system. In contrast, the promoters of dm-I, which shows induced expression, and cf-I, which has constitutive expression, were not methylated in any organs examined. Thus, it seems that the regulatory mechanisms of the da-I, dm-I and cf-I genes differ from each other and do not correlate with the phylogenetic relationship. The current study suggests that under environmental pressure in specific habitats carnivorous plants have managed to evolve their S-like RNase genes to function in carnivory.

Glucan-rich diet is digested and taken up by the carnivorous sundew (Drosera rotundifolia L.): implication for a novel role of plant ß-1,3-glucanases.

Carnivory in plants evolved as an adaptation strategy to nutrient-poor environments. Thanks to specialized traps, carnivorous plants can gain nutrients from various heterotrophic sources such as small insects. Digestion in traps requires a coordinated action of several hydrolytic enzymes that break down complex substances into simple absorbable nutrients. Among these, several pathogenesis-related proteins including ß-1,3-glucanases have previously been identified in digestive fluid of some carnivorous species. Here we show that a single acidic endo-ß-1,3-glucanase of ~50 kDa is present in the digestive fluid of the flypaper-trapped sundew (Drosera rotundifolia L.). The enzyme is inducible with a complex plant ß-glucan laminarin from which it releases simple saccharides when supplied to leaves as a substrate. Moreover, thin-layer chromatography of digestive exudates showed that the simplest degradation products (especially glucose) are taken up by the leaves. These results for the first time point on involvement of ß-1,3-glucanases in digestion of carnivorous plants and demonstrate the uptake of saccharide-based compounds by traps. Such a strategy could enable the plant to utilize other types of nutritional sources e.g., pollen grains, fungal spores or detritus from environment. Possible multiple roles of ß-1,3-glucanases in the digestive fluid of carnivorous sundew are also discussed.

Detection of chitinolytic enzymes with different substrate specificity in tissues of intact sundew (Drosera rotundifolia L.): chitinases in sundew tissues.

The round-leaf sundew (Drosera rotundifolia L.) is a carnivorous plant expressing a wide range of chitinolytic enzymes playing role in many different processes. In this study the intact plants were analyzed for the presence of chitinase transcripts and chitinolytic activities in different organs. In situ hybridization with chitnase fragment as a probe has revealed the presence of chitinases in the mesophyll cells of leaves and vascular elements of stems of healthy, non-stressed plants. More pronounced expression was observed in cortex and stele cells of roots as well as in ovules and anthers of reproductive organs. Similarly, higher chitinase enzyme activity was typical for flowers and roots suggesting a more specific role of chitinases in these tissues. In addition to endochitinases of different substrate specificities, chitobiosidases contributed to overall chitinolytic activity of tissue extracts. The activity of chitobiosidases was again typical for flowers and roots, while their role in plant physiology remains to be elucidated.

An S-like ribonuclease gene is used to generate a trap-leaf enzyme in the carnivorous plant Drosera adelae.

Carnivorous plants usually grow in nutrient-deficient habitats, and thus they partly depend on insects for nitrogen and phosphate needed for amino acid and nucleotide synthesis. We report that a sticky digestive liquid from a sundew, Drosera adelae, contains an abundant amount of an S-like ribonuclease (RNase) that shows high amino acid-sequence similarity to S-like RNases induced by phosphate starvation or wounding in normal plants. By giving leaves an RNase "coat", D. adelae seems to achieve two requirements simultaneously to adapt itself to its specific surroundings: it obtains phosphates from insects, and defends itself against pathogen attack.

Tentacles of in vitro-grown round-leaf sundew (Drosera rotundifolia L.) show induction of chitinase activity upon mimicking the presence of prey.

Induction of plant-derived chitinases in the leaves of a carnivorous plant was demonstrated using aseptically grown round-leaf sundew (Drosera rotundifolia L.). The presence of insect prey was mimicked by placing the chemical inducers gelatine, salicylic acid and crustacean chitin on leaves. In addition, mechanical stirring of tentacles was performed. Chitinase activity was markedly increased in leaf exudates upon application of notably chitin. Application of gelatine increased the proteolytic activity of leaf exudates, indicating that the reaction of sundew leaves depends on the molecular nature of the inducer applied. In situ hybridization of sundew leaves with a Drosera chitinase probe showed chitinase gene expression in different cell types of non-treated leaves, but not in the secretory cells of the glandular heads. Upon induction, chitinase mRNA was also present in the secretory cells of the sundew leaf. The combined results indicate that chitinase is likely to be involved in the decomposition of insect prey by carnivorous plants. This adds a novel role to the already broad function of chitinases in the plant kingdom and may contribute to our understanding of the molecular mechanisms behind the ecological success of carnivorous plants in nutritionally poor environments.