Discovery of digestive enzymes in carnivorous plants with focus on proteases

Introduction

Nitrogen is the most crucial mineral nutrient required by plants but its availability is largely limited in many terrestrial ecosystems (Behie & Bidochka, 2013). For adaptation to such unfavourable environment, carnivorous plants have developed the ability to attract, capture, and digest preys into simpler mineral compounds, which are then absorbed for plant growth and reproduction (Ellison, 2006). The first evidence on the ability of the plant to capture and digest insects was provided over 140 years ago (Darwin, 1875). Since then, more than 700 carnivorous species from 20 genera of 12 families (Givnish, 2015) have been identified with captivating morphological and physiological traits linked to carnivory (Król et al., 2011).

There are a few reviews on the evolution of carnivorous plants and their biotechnological applications (Król et al., 2011; Miguel, Hehn & Bourgaud, 2018). However, a systematic review with focus on digestive enzyme discovery and characterisation from all families of carnivorous plants is lacking. Furthermore, the pharmacological potentials of some of these carnivorous plants have also been largely overlooked. With the advent of omics technology which accelerated enzyme discovery in carnivorous plants for the past few years, there is a pressing need for a timely review on current progress of studies in this field. This review will be useful not only to researchers working on carnivorous plants, but also those with interest in commercially useful enzymes and natural products.

Survey Methodology

In this review, we provide perspectives on the latest research of different carnivorous plants, namely Cephalotus, Drosera, Dionaea, Genlisea, Nepenthes, Sarracenia, and Utricularia, on their digestive enzyme discovery and characterisation. In earlier studies, research interest on carnivorous plants was centred on axenic culture, ultrastructure of specialised trapping organs, foliar absorption of nutrients derived from preys, and the enzymatic studies of prey digestion (Adamec, 1997; Gorb et al., 2004; Farnsworth & Ellison, 2008). Thus, this review summarises the previous findings with focus on digestive enzymes discovered in carnivorous plants, especially proteases and their industrial applications. The literature survey was performed exhaustively online using Google search engine and SCOPUS. The discussion will be mainly based on recent studies.

Digestive Enzyme Discovery, Identification and Characterisation

Digestion of prey in carnivorous plants relies on enzymes which could be associated with morphologically diverse trapping mechanisms. There are a few studies which reported that the secretion of the digestive enzymes is strongly induced by prey capture. However, there are also certain digestive enzymes which are readily secreted in the absence of prey. This indicates plant regulation of enzyme secretion because the production and secretion of enzymes incur energetic costs.

To date, numerous studies had reported the discovery of distinct digestive enzymes in carnivorous plants (Table 2). Similar enzymes with various enzymatic properties were shared among different carnivorous families. With the genome sequencing of Cephalotus follicularis, various digestive enzymes were discovered, namely esterases, proteases, nucleases, phosphatases, glucanases, and peroxidases (Takahashi et al., 2009; Fukushima et al., 2017). Similar classes of enzymes were also detected in other carnivorous families, such as Droseraceae, Lentibulariacea, Sarraceniaceae, and Nepenthaceae. This suggests significant role of common hydrolytic enzymes, especially phosphatases, proteases, and chitinases, in prey digestion of various carnivorous plants regardless of different families or trapping mechanisms. Recently, Yilamujiang et al. (2017) reported the presence of a novel digestive enzyme urease in N. hemsleyana which has developed a symbiosis relationship with bat.

However, investigation related to the identification of proteins found in the pitcher fluid is highly challenged by unusual amino acid composition and limited carnivorous plant genome or protein sequence database (Lee et al., 2016). Early study by Amagase (1972) utilised zymography technique to determine the protease activity found in fluid of Nepenthes spp. and D. peltata. The fluids were purified and characterised for acid protease and demonstrated similar protease activity from two distinct families. Later, Hatano & Hamada (2008) conducted proteomic analysis on the digestive fluid of N. alata in which secreted chitinase, glucanase, and xylosidase were identified through in-gel trypsin digestion, de novo peptide assembly, and homology search using public databases. Recently, a transcriptomic approach was taken for N. ampullaria, N. rafflesiana, N. × hookeriana, and N. × ventrata (Wan Zakaria et al., 2016a; Wan Zakaria et al., 2016b; Zulkapli et al., 2017), which can serve as reference sequences for identifying more digestive enzymes through proteomics analysis (Wan Zakaria et al., 2018). A proteomics informed by transcriptomics approach was taken by Schulze et al. (2012) to determine the proteins highly expressed in the digestive fluid of Venus flytrap. They discovered a coordinated prey digestion mechanism facilitated by various enzymes, such as chitinases, lipases, phosphatases, peroxidases, glucanases, and peptidases. Fluorescent resonance energy transfer (FRET) based technique can be utilised as an efficient and rapid detection of proteolytic activities in the pitcher fluid of various Nepenthes species (Buch et al., 2015). Rey et al. (2016) applied a similar approach to assess proteolytic efficiency of the protein secreted in the pitcher fluid of Nepenthes species.

On the other hand, purification of digestive enzymes from carnivorous fluid is extremely challenging due to low amount of secreted fluid and enzyme. Furthermore, pitcher fluids are often diluted with rainwater and even contaminated by decomposing prey. Nevertheless, there are studies which manage to purify and characterise digestive enzymes from carnivorous plants (Table 3). Based on the reported purification and characterisation studies, proteases are the most abundant enzymes characterised from the digestive fluid of carnivorous plant. The very first purification of protease from pitcher fluid of Nepenthes species was performed by Steckelberg, Lüttge & Weigl (1967) using Ecteola column chromatography and its optimum activity was detected at pH 2.2 with stability at 50 °C. To date, the common purification strategies applied by various studies are column chromatography, affinity chromatography, ultrafiltration, and dialysis. Although many digestive enzymes have been identified from carnivorous plants, only few studies have purified and characterised the enzymes. Therefore, further studies on the purification and characterisation of various digestive enzymes are needed.

Most of the characterised enzymes can catalyse various substrates and activities of the same category of enzymes from different carnivorous plants are similar in terms of optimum pH, temperature, and substrate specificity (Table 3). For instance, most of the characterised proteases from different families function optimally at acidic condition. Interestingly, there are a few proteases reported to function optimally at high temperature ranging from 40−60 °C. Additionally, the secreted enzymes demonstrate higher stability against various chemicals and denaturing agents than similar enzymes from other sources. This is because prey digestion often occurs over long period under varied conditions, thus digestive enzymes are important to be active and stable (Butts, Bierma & Martin, 2016). Subtle variations in enzymatic characteristics of digestive enzymes from different carnivorous plants remain to be explored. Furthermore, nomenclature of enzymes reported from different carnivorous plants need to be standardised for comparative studies.

There are only a few reports on the structural characterisation of the digestive enzymes secreted by carnivorous plants. To date, proteases and chitinases are the most characterised in structural and enzymatic properties (Ishisaki et al., 2012a; Fukushima et al., 2017; Jopcik et al., 2017; Unhelkar et al., 2017). Athauda et al. (2004) was the first to report a complete model of purified Nepenthesin from N. distillatoria. Interestingly, nepenthesin contains extra three disulphide bonds in the N-terminal compared to only three disulphide bonds in porcine pepsin A (Fig. 1). Comparison of predicted protease structures of Nepenthesin I and Nepenthesin II from N. alata show similarities in the location of catalytic Asp residues. Nepenthesin is distinct from pepsin with a nepenthesin-type aspartic protease (NAP)-specific insert with four conserved cysteine residues believed to confer higher protein stability. Further structural analysis on proteases from carnivorous plants can refer to a recent study by Butts, Bierma & Martin (2016).

On the other hand, feeding with insect or chitin induces the secretion of enzymes in digestive fluid. Clancy & Coffey (1977) have reported the maximal secretion of digestive enzymes, specifically phosphatases and proteases in Venus flytrap and Drosera within 3 to 4 days after feeding. Apart from that, mechanical irritation also stimulates the increase in the activity of phosphatases and phosphodiesterases in Drosera (Mcnally, Stewart & Wilson, 1988). Moreover, the quantity of enzymes secreted often associates with the size of prey (Darwin, 1875; An, Fukusaki & Kobayashi, 2002). These reports suggest a signal transduction mechanism which stimulates the expression of digestive enzymes, allowing plants to respond accordingly toward prey for optimal cost-benefit ratio (Chang & Gallie, 1997).

The origin of enzymes found in digestive fluid has been controversial as to whether all are plant secreted or derived from microbial community found in the digestive fluid. A study reported high expression of hydrolytic enzymes in the digestive zone of pitcher trap (An, Fukusaki & Kobayashi, 2002). Meanwhile, a study on Sarracenia pitcher showed there is a symbiotic interaction between microbial community in the pitcher fluid and the plant in prey digestion (Koopman et al., 2010). This study suggests that some carnivorous plants could be co-opting microbes for initial prey digestion and secrete digestive enzymes for later stage of digestion. From a different perspective, prey digestion through plant enzymes could be enhanced through symbiotic relationship with microbes or fungi to decompose prey into simpler form of nutrients. This mutualistic interaction with microbial community in the digestive fluid will boost digestion and nutrient absorption. However, there must be a balancing point or even selection of microbial community (Takeuchi et al., 2015) to prevent competitive loss of nutrients as indicated by various defence-related proteins (Lee et al., 2016; Rottloff et al., 2016) and antimicrobial naphthoquinones (Buch et al., 2012) found in the pitcher fluid.

Secreted Proteases in Different Families of Carnivorous Plants

Carnivorous plants attain substantial amount of nitrogen from prey through specialised trapping organs which accumulate acidic fluid containing protease. Early reports of digestive enzymes involved in carnivorous plants were initiated by Sir Joseph Hooker’s studies of protease activity in the pitcher fluid of Nepenthes plants (Renner & Specht, 2013). Independent evolution of carnivorous plants might have resulted in convergent evolution of diverse digestive enzymes serving similar functions (Fukushima et al., 2017).

Aspartic proteases (APs), such as nepenthesin, are one of the most abundant and well characterised enzymes found in the digestive fluid (An, Fukusaki & Kobayashi, 2002; Rottloff et al., 2016). AP have been purified and characterised from sterile pitcher fluid of several Nepenthes species (Jentsch, 1972; Tokes, Woon & Chambers, 1974). In a study conducted by Nakayama & Amagase (1968), a protease from pooled pitcher fluids of N. mixta and N. maxima was only partially purified and characterised due to insufficient amount. Amagase (1972) investigated aspartic proteases found in N. ampullaria, N. mixta, N. rafflesiana, N. maxima, and N. dyeriana compared to leaf extract from Drosera peltata. Lately, acid protease from Nepenthes and Drosera genus are partially purified and characterised (Takahashi, Tanji & Shibata, 2007; Tokes, Woon & Chambers, 1974). Surprisingly, both the purified proteases from Nepenthes and Drosera share common characteristics. An, Fukusaki & Kobayashi (2002) cloned homologous AP genes and examined their expression in N. alata. The protease secreted in the pitcher fluid is pepsin-like and active at acidic condition (Rudenskaya et al., 1995). Although they have been categorised as APs, none of the native enzymes was purified to homogeneity, mainly due to difficulty in obtaining sufficient amount of pitcher fluid. Later, Athauda et al. (2004) for the first time purified and characterised two APs, namely Nep1 and Nep2, from pitcher fluid N. distillatoria. They also characterised the amino acid sequences of the enzymes by cloning the cDNAs from pitcher tissue of N. gracilis. Recently, five nepenthesins were reported to be secreted in Nepenthes pitcher fluid (Lee et al., 2016). However, little is known about why there are various AP genes expressed in Nepenthes pitcher fluid and their differential regulations if any. It is key to a better understanding of the regulation of nitrogen-acquisition mechanism in Nepenthes plants.

Apart from aspartic proteases, there is also presence of cysteine proteases in carnivorous plants. Lately, it also has been found that cysteine protease is the primary protease found in digestive fluid of Dionaea (Venus flytrap). Prey proteins found in the digestive fluid of Dionaea are degraded by cysteine endopeptidases in association with serine carboxypeptidases (Risør et al., 2016). This is highly distinct to the digestive fluids found in Nepenthes and Drosera with aspartic proteases (Athauda et al., 2004). However, there is also the presence of both aspartic and cysteine proteases in N. ventricosa as reported by Stephenson & Hogan (2006). Takahashi, Tanji & Shibata (2007) conducted comparative enzymatic characterisation of acid proteases from crude digestive fluid of various carnivorous plants namely Nepenthes, Chepalotus, Drosera, and Dionaea, with distinct trapping mechanisms. The study demonstrated significant variations between them, which might be due to the presence of different classes of proteases in different families. This reflects the phylogenetic diversity of these carnivorous plants.

There are attempts on the recombinant expression of the enzymes from carnivorous plants (Morohoshi et al., 2011; Ishisaki et al., 2012b; Kadek et al., 2014b). Kadek et al. (2014b) reported an efficient way to obtain high amount of Nepenthesin I (Nep1) from N. gracilis through heterologous expression in Escherichia coli. The characteristics of the recombinant protein obtained are similar to the native enzyme isolated from the pitcher fluid. More recently, Nep1 from N. gracilis was successfully purified and crystallised (Fejfarová et al., 2016).

On the other hand, the evolution of different trapping mechanisms for carnivorous plants to survive in harsh environments with limited nutrients may result in enzymes with novel properties. For instance, a novel class of prolyl endopeptidase called neprosin 1 and neprosin 2 (Npr1 & Npr2) was recently discovered in Nepenthes species to be distinct from commonly known proline-cleaving enzymes, which consists of two novel neprosin domains (Lee et al., 2016). Schrader et al. (2017) characterised neprosin to be a proline-cleaving enzyme through recombinant approach and demonstrated that it has the potential to be utilised for whole proteomic profiling and histone mapping. This is because neprosin is a low molecular weight prolyl endopeptidase and extremely active at low concentration and pH. Combined actions of a neprosin and nepenthesin from Nepenthes pitcher fluid showed potential of effective gluten detoxification, which broaden the prospects for enzyme supplementation approach to circumvent celiac disease (Rey et al., 2016).

Although the proteolytic activity in the digestive fluid is of great interest, low yields of secreted enzymes make it very challenging for native enzyme purification. Furthermore, prey digestion is likely to be concerted activities of various proteases and other enzymes in the digestive fluid, hence it is interesting to compare the enzyme assays between crude digestive fluid extracts and individual purified proteases.

Conclusions

The search for new industrially viable plant enzymes is a continuous effort in which carnivorous plants serve as great resources for exploration. There are numerous studies on the properties of digestive fluid of carnivorous plants that contribute to a better understanding of carnivory mechanism and evolution. Further extensive biochemical and morphological studies on carnivorous plants will still be needed to help in further understanding the regulation of hydrolytic enzyme secretion. In addition, successful purification and characterisation of the secreted enzymes will encourage their exploitation for industrial applications. Future research efforts in studying regulatory mechanisms of digestive enzymes or metabolites responsible for attracting prey will not only be useful to fill in current gaps in knowledge, but also advancing novel utilisation of carnivorous plants for producing plant-made proteins. Comparative genomics approach will help in elucidating the evolutionary history of these fascinating plants. With the advent of omics technologies, a holistic understanding on the molecular mechanisms of carnivory in various carnivorous plants will be achievable along with more exciting discoveries.