Adaptations to foliar absorption of faeces: a pathway in plant carnivory

BACKGROUND AND AIMS: Roridula plants capture insects but have no digestive enzymes. It has been hypothesized that Roridula leaves absorb nitrogen from the faeces of obligately associated, carnivorous hemipterans. But rapid movement across the leaf surfaces of most plant leaves is prevented by the presence of an impermeable cuticle. However, in carnivorous plants, cuticular gaps or pores in digestive/absorptive cells allow rapid movement across the leaf surface. Recently, it was suggested that the hemipteran-plant interaction constituted a new pathway for plant carnivory. Here, a further adaptation to this pathway is described by demonstrating how Roridula plants probably absorb hemipteran faeces rapidly through their leaf cuticles. METHODS: The dye neutral red was used to document the rapidity of foliar absorption and TEM to determine the nature of cuticular discontinuities in the leaf of Roridula. KEY RESULTS: Aqueous compounds diffuse rapidly across the cuticle of Roridula's leaves but not across the cuticles of co-occurring, non-carnivorous plant leaves. Furthermore, immature Roridula leaves were unable to absorb neutral red whereas mature leaves could. Using TEM, cuticular gaps and pores similar to those in other carnivorous plants were found in the epidermal cells of mature Roridula leaves. CONCLUSIONS: The leaf cuticle of Roridula is very thin (0-120 nm) and cell wall elements project close to the leaf surface, possibly enhancing foliar absorption. In addition to these, cuticular gaps were frequently seen and probably perform a function similar to those found in other carnivorous plants: namely the absorption of aqueous compounds. The cuticular gaps of Roridula are probably an adaptation to plant carnivory, supporting the newly described pathway.     

Glucan-rich diet is digested and taken up by the carnivorous sundew (Drosera rotundifolia L.): implication for a novel role of plant β-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 β-1,3-glucanases have previously been identified in digestive fluid of some carnivorous species. Here we show that a single acidic endo-β-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 β-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 β-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 β-1,3-glucanases in the digestive fluid of carnivorous sundew are also discussed.     

The Ultrastructure of the Digestive Glands in Pinguicula vulgaris L. (Lentibulariaceae) Relative to their Function. II. The Changes on Stimulation

The secretory cells of the digestive glands remain highly activeduring the entire period of prey digestion and absorption ofnutrients. They appear to play a major role in gland activity.A model of the digestive gland's activity on stimulation isproposed. It is very similar to that suggested earlier for Dionaeamuscipula. After the digestion and absorption cycle, destructiveprocesses are initiated in the glands. These appear similarto those observed in the glands of the ageing, unstimulatedleaf and are not associated with feeding. Pinguicula vulgaris L. carnivorous plant, digestive glands, ultrastructure, protein secretion absorption, senescence     

Carnivorous syndrome in Asian pitcher plants of the genus Nepenthes

BACKGROUND AND AIMS: Pitcher plants Nepenthes alata and N. mirabilis are carnivorous species with leaves composed of a photosynthetic part (lamina) and a pitcher trap. This characteristic permitted direct physiological and anatomical comparison between these two distinct parts of the leaves to determine those features involved in the 'carnivorous syndrome', which include low net photosynthetic assimilation rate (A(N)) and low photosynthetic nitrogen use efficiency (PNUE). METHODS: Photosynthetic rate (A(N)) and respiration rate (R(d)) were measured gasometrically, chlorophyll concentration was determined spectrophotometrically and nitrogen concentration was determined using a CHN elemental analyser in lamina and trap separately. Anatomy of N. alata was observed using light, fluorescence and transmission electron microscopy. A(N), foliar nitrogen and chlorophyll concentration were also compared with values for other carnivorous plant species (genera Sarracenia, Drosera) that combine both autotrophic and carnivorous functions into the same physical organ. KEY RESULTS: It was found that the A(N) in Nepenthes lamina was low and PNUE was only slightly higher or similar in comparison with other carnivorous plants. It was not observed that the pitcher had a higher R(d) than the lamina, but A(N) in the pitcher was significantly lower than in the lamina. Nepenthes possesses a cluster of characters that could result in reduced photosynthesis in the pitcher and be responsible for carnivorous function of the leaf: replacement of chlorophyll-containing cells with digestive glands, low chlorophyll and nitrogen concentration, compact mesophyll with a small portion of intercellular spaces, absence of palisade parenchyma and low stomatal density. CONCLUSION: Low photosynthetic capacity, nitrogen efficiency, chlorophyll and nitrogen concentration of Nepenthes pitchers was found, together with a set of features that characterized the carnivorous syndrome. Dual use of leaves for photosynthesis and nutrient gain can decrease photosynthetic efficiency in carnivorous plants in general.     

The Ultrastructure of the Digestive Glands in Pinguicula vulgaris L. (Lentibulariaceae) Relative to their Function. I. The Changes During Maturation

The digestive glands of Pinguicula vulgaris become fully maturewhilst still enclosed in the bud. All the gland cells remainintact on the fully expanded unstimulated leaves. As the secretoryhead cells mature, a special layer forms between the plasmalemmaand the cell wall. This layer is shown to be different fromthe typical labyrinthine wall of transfer cells and serves forthe storage of digestive enzymes. Ultrastructural analysis,including morphometry, indicates that the digestive enzymesare synthesized on the RER of the head cells and transferredinto the cell wall, particularly into the slime layer, and vacuoles.This transfer is achieved firstly through continuity of theendoplasmic reticulum with vacuoles (static) and the periplasmicspace (dynamic) and, secondly, into the latter through exocytosisof coated Golgi vesicles and of some vacuoles filled with enzymes. Pinguicula vulgaris L., carnivorous plant, digestive glands, ultrastructure, protein synthesis secretion     

Pathways for Nutrient Transport in the Pitchers of the Carnivorous Plant Nepenthes alata

Glands of the carnivorous pitcher plant Nepenthesalata are activein transport of materials into and out of the pitcher lumen,indicating dual functions in both secretion and absorption.This study examined the potential for open transport throughthese glands using the ultrastructural tracer lanthanum, whichis restricted to the apoplast, and the fluorescent symplastictracer, 6(5)carboxyfluorescein. Glandular uptake of lanthanumfrom the pitcher fluid occurred through the outer cell wallbetween irregularly spaced cutinized deposits, but was blockedfrom entering the underlying mesophyll cell walls by thick endodermal-likeregions. Similarly, lanthanum localization showed an open apoplasticpathway from the petiole to the endodermal regions in the glandbase. Thus, transport of materials into or out of the glandmust occur through the symplast. 6(5)Carboxyfluorescein showedthat these glands transport fluids directly from the pitcherfluid into vascular endings immediately beneath them via a symplasticroute. When applied to the petiolar vascular system, the fluorescenttracer freely entered immature pitchers, but was blocked fromentering the lumen of the mature pitcher by an endodermal zone.An ultrastructural survey showed infrequent pits with plasmodesmatalconnections to adjoining subepidermal cells. These results indicatethat the function of the gland is developmentally regulated.Prior to maturity, the primary function of the gland appearsto be secretion. However, at maturity, secretion is blockedby an endodermal layer, which limits the function of the glandto absorption. These studies support the theory that the glandsof Nepenthesalata are specialized for the bi-directional transportof materials.Copyright 1999 Annals of Botany Company Apoplastic transport, 6(5)carboxyfluorescein, carnivorous plants, digestive glands, endodermal layer,Nepenthesalata Blanco, lanthanum, pitcher plants.     

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant,Nepenthes

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. These structures are almost always localized on plant surfaces. Among them are nectaries or glandular trichomes. The secreted compounds are often employed in interactions with the biotic environment, for example as attractants for pollinators or deterrents against herbivores.Glands that are unique in several aspects can be found in carnivorous plants. In so-called pitcher plants of the genus Nepenthes, bifunctional glands inside the pitfall-trap on the one hand secrete the digestive fluid, including all enzymes necessary for prey digestion, and on the other hand take-up the released nutrients. Thus, these glands represent an ideal, specialized tissue predestinated to study the underlying molecular, biochemical, and physiological mechanisms of protein secretion and nutrient uptake in plants. Moreover, generally the biosynthesis of secondary compounds produced by many plants equipped with glandular structures could be investigated directly in glands.In order to work on such specialized structures, they need to be isolated efficiently, fast, metabolically active, and without contamination with other tissues. Therefore, a mechanical micropreparation technique was developed and applied for studies on Nepenthes digestion fluid. Here, a protocol is presented that was used to successfully prepare single bifunctional glands from Nepenthes traps, based on a mechanized microsampling platform. The glands could be isolated and directly used further for gene expression analysis by PCR techniques after preparation of RNA.     

Molecular and functional evolution of class I chitinases for plant carnivory in the caryophyllales

Proteins produced by the large and diverse chitinase gene family are involved in the hydrolyzation of glycosidic bonds in chitin, a polymer of N-acetylglucosamines. In flowering plants, class I chitinases are important pathogenesis-related proteins, functioning in the determent of herbivory and pathogen attack by acting on insect exoskeletons and fungal cell walls. Within the carnivorous plants, two subclasses of class I chitinases have been identified to play a role in the digestion of prey. Members of these two subclasses, depending on the presence or absence of a C-terminal extension, can be secreted from specialized digestive glands found within the morphologically diverse traps that develop from carnivorous plant leaves. The degree of homology among carnivorous plant class I chitinases and the method by which these enzymes have been adapted for the carnivorous habit has yet to be elucidated. This study focuses on understanding the evolution of carnivory and chitinase genes in one of the major groups of plants that has evolved the carnivorous habit: the Caryophyllales. We recover novel class I chitinase homologs from species of genera Ancistrocladus, Dionaea, Drosera, Nepenthes, and Triphyophyllum, while also confirming the presence of two subclasses of class I chitinases based upon sequence homology and phylogenetic affinity to class I chitinases available from sequenced angiosperm genomes. We further detect residues under positive selection and reveal substitutions specific to carnivorous plant class I chitinases. These substitutions may confer functional differences as indicated by protein structure homology modeling.     

Rapid responses: Receptor-like kinases directly regulate the functions of membrane transport proteins in plants

Receptor-like kinases (RLKs) are a large group of plant-specific transmembrane proteins mainly acting as receptors or co-receptors of various extracellular signals. They usually turn extracellular signals into intracellular responses via altering gene expression profiles. However, recent studies confirmed that many RLKs can physically interact with diverse membrane-localized transport proteins and regulate their activities for speedy responses in limited tissues or cells. In this minireview, we highlight recent discoveries regarding how RLKs can work with membrane transport proteins collaboratively and thereby trigger cellular responses in a precise and rapid manner. It is anticipated that such regulation broadly presents in plants and more examples will be gradually revealed when in-depth analyses are conducted for the functions of RLKs.     

A novel insight into the cost–benefit model for the evolution of botanical carnivory

Background The cost–benefit model for the evolution of botanical carnivory provides a conceptual framework for interpreting a wide range of comparative and experimental studies on carnivorous plants. This model assumes that the modified leaves called traps represent a significant cost for the plant, and this cost is outweighed by the benefits from increased nutrient uptake from prey, in terms of enhancing the rate of photosynthesis per unit leaf mass or area (A_N) in the microsites inhabited by carnivorous plants.Scope This review summarizes results from the classical interpretation of the cost–benefit model for evolution of botanical carnivory and highlights the costs and benefits of active trapping mechanisms, including water pumping, electrical signalling and accumulation of jasmonates. Novel alternative sequestration strategies (utilization of leaf litter and faeces) in carnivorous plants are also discussed in the context of the cost–benefit model.Conclusions Traps of carnivorous plants have lower A_N than leaves, and the leaves have higher A_N after feeding. Prey digestion, water pumping and electrical signalling represent a significant carbon cost (as an increased rate of respiration, R_D) for carnivorous plants. On the other hand, jasmonate accumulation during the digestive period and reprogramming of gene expression from growth and photosynthesis to prey digestion optimizes enzyme production in comparison with constitutive secretion. This inducibility may have evolved as a cost-saving strategy beneficial for carnivorous plants. The similarities between plant defence mechanisms and botanical carnivory are highlighted.     

The Roots of Carnivorous Plants

Carnivorous plants may benefit from animal-derived nutrients to supplement minerals from the soil. Therefore, the role and importance of their roots is a matter of debate. Aquatic carnivorous species lack roots completely, and many hygrophytic and epiphytic carnivorous species only have a weakly devel-oped root system. In xerophytes, however, large, extended and/or deep-reaching roots and sub-soil shoots develop. Roots develop also in carnivorous plants in other habitats that are hostile, due to flood-ing, salinity or heavy metal occurance. Information about the structure and functioning of roots of car- nivorous plants is limited, but this knowledge is essential for a sound understanding of the plants’ physiology and ecology. Here we compile and summarise available information on: (1) The morphology of the roots. (2) The root functions that are taken over by stems and leaves in species without roots or with poorly developed root systems; anchoring and storage occur by specialized chlorophyll-less stems; water and nutrients are taken up by the trap leaves. (3) The contribution of the roots to the nutrient supply of the plants; this varies considerably amongst the few investigated species. We compare nutrient uptake by the roots with the acquisition of nutri-ents via the traps. (4) The ability of the roots of some carnivorous species to tolerate stressful conditions in their habitats; e.g., lack of oxygen, saline conditions, heavy metals in the soil, heat during bushfires, drought, and flooding     

An experimental test of the defensive role of sticky traps in the carnivorous plant Pinguicula moranensis (Lentibulariaceae)

It has been sustained that the sticky traps present in some carnivorous plants could have evolved from ancestor species bearing leaves covered with secreting glands formerly associated with a defensive function. In this study, we evaluated the interaction of the carnivorous plant Pinguicula moranensis with its insect herbivores to assess the defensive role of the glandular trichomes. Firstly, we estimated the standing levels of insect herbivory in field conditions. We also evaluated the response of herbivore insects to the removal of the secreting glands from the leaves of P. moranensis in field and laboratory conditions. The mean damage was 1.61%, and half of the sampled plants showed no damage. The low level of herbivory in the field suggests that P. moranensis has an efficient defense ability. In the field experiment, after 25 d of exposure to natural damage, treated glandless plants received 18 times more damage than control plants. In the laboratory, the consumption of glandless tissue was three times higher during a 6 h evaluation period. Overall, our results provide evidence that secreting trichomes in Pinguicula are not only associated with prey capture but also have a defensive role. The defensive function could have favored the evolution of the sticky traps, the most extended prey‐capture strategy among carnivorous plants.     

Recent ecophysiological,biochemical and evolutional insights into plant carnivory

BackgroundCarnivorous plants are an ecological group of approx. 810 vascular species which capture and digest animal prey, absorb prey-derived nutrients and utilize them to enhance their growth and development. Extant carnivorous plants have evolved in at least ten independent lineages, and their adaptive traits represent an example of structural and functional convergence. Plant carnivory is a result of complex adaptations to mostly nutrient-poor, wet and sunny habitats when the benefits of carnivory exceed the costs. With a boost in interest and extensive research in recent years, many aspects of these adaptations have been clarified (at least partly), but many remain unknown.ScopeWe provide some of the most recent insights into substantial ecophysiological, biochemical and evolutional particulars of plant carnivory from the functional viewpoint. We focus on those processes and traits in carnivorous plants associated with their ecological characterization, mineral nutrition, cost–benefit relationships, functioning of digestive enzymes and regulation of the hunting cycle in traps. We elucidate mechanisms by which uptake of prey-derived nutrients leads to stimulation of photosynthesis and root nutrient uptake.ConclusionsUtilization of prey-derived mineral (mainly N and P) and organic nutrients is highly beneficial for plants and increases the photosynthetic rate in leaves as a prerequisite for faster plant growth. Whole-genome and tandem gene duplications brought gene material for diversification into carnivorous functions and enabled recruitment of defence-related genes. Possible mechanisms for the evolution of digestive enzymes are summarized, and a comprehensive picture on the biochemistry and regulation of prey decomposition and prey-derived nutrient uptake is provided.     

Sticky plant captures prey for symbiotic bug: is this digestive mutualism?

Many plants capture and kill insects but, until relatively recently, only carnivorous plants with digestive enzymes were known to gain directly from the nutrients of those insects. Recent studies show that some carnivorous plants lack digestive enzymes and have evolved digestive mutualisms with symbiotic insects that digest their prey for them. Rhododendron macrosepalum, a plant with sticky leaves that captures insects, has an association with symbiotic Mirid bugs that consume the insects captured. Here, we determine what the nature of the relationship is between Mirid and plant. We find that R. macrosepalum has no digestive enzymes of its own but that it does not seem to have the ability to absorb hemipteran faeces through its leaf cuticle. Naturally occurring levels of ¹⁵N and ¹⁴N were used to determine that R. macrosepalum gains no nitrogen through its association with the Mirid bugs and that it obtains all of its nitrogen from the soil. The Mirids, on the other hand, seem to obtain nitrogen from insects captured by the plant, as well as from plant tissues. The relationship between plant and Mirid is not a digestive mutualism but more likely an antagonistic relationship. This study adds to our understanding of how digestive mutualisms evolve and shows that insect capture alone, or in combination with a symbiotic insect relationship does not necessarily make a plant ‘carnivorous’.     

Endocytotic uptake of nutrients in carnivorous plants

Carnivorous plants trap, digest and absorb animals in order to supplement their mineral nutrition. Nutrients absorbed by the plant include different nitrogen species, phosphate, potassium, trace elements and small organic compounds. Uptake is usually thought to be performed via specific channels, but this study provides evidence that endocytosis is involved as well. Traps of the carnivorous plants Nepenthes coccinea, Nepenthes ventrata, Cephalotus follicularis, Drosophyllum lusitanicum, Drosera capensis, Dionaea muscipula, Aldrovanda vesiculosa, Genlisea violacea × lobata, Sarracenia psittacina and Sarracenia purpurea were stained with methylene blue in order to identify possible sites of uptake. The permeable parts of the traps were incubated with fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA) and other fluorescent endocytosis markers, combined with the soluble protein BSA or respiratory inhibitors. Uptake was studied by confocal microscopy. In Nepenthes, small fluorescent vesicles became visible 1 h after incubation with FITC-BSA. These vesicles fused to larger compartments within 30 h. A similar behaviour was found in the related genera Drosera, Dionaea, Aldrovanda and Drosophyllum but also in Cephalotus with glands of different evolutionary origin. In Genlisea and Sarracenia, no evidence for endocytosis was found. We propose that in many carnivorous plants, nutrient uptake by carriers is supplemented by endocytosis, which enables absorption and intracellular digestion of whole proteins. The advantage for the plant of reducing secretion of enzymes for extracellular digestion is evident.     

Cytochemical and ultrastructural aspects of aquatic carnivorous plant turions

Turions, which are modified shoot apices, are vegetative, dormant overwintering organs produced by perennial aquatic plants. In this study, the turion cytochemistry and ultrastructure of Aldrovanda vesiculosa, Utricularia vulgaris and U. stygia were compared with particular emphasis placed on storage substances. These three aquatic, rootless carnivorous plant species were studied at the end of their winter dormancy. At this stage, the turions of all species had starch as their main storage material. In contrast with A. vesiculosa, Utricularia turions were rich in protein storage vacuoles, and proteins were also accumulated as crystalline inclusions in the nuclei. All examined species accumulated lipid droplets in cells of epidermal glands.     

Fluorescence labelling of phosphatase activity in digestive glands of carnivorous plants

Abstract: A new ELF (enzyme labelled fluorescence) assay was applied to detect phosphatase activity in glandular structures of 47 carnivorous plant species, especially Lentibulariaceae, in order to understand their digestive activities. We address the following questions: (1) Are phosphatases produced by the plants and/or by inhabitants of the traps? (2) Which type of hairs/glands is involved in the production of phosphatases? (3) Is this phosphatase production a common feature among carnivorous plants or is it restricted to evolutionarily advanced species? Our results showed activity of the phosphatases in glandular structures of the majority of the plants tested, both from the greenhouse and from sterile culture. In addition, extracellular phosphatases can also be produced by trap inhabitants. In Utricularia, activity of phosphatase was detected in internal glands of 27 species from both primitive and advanced sections and different ecological groups. Further positive reactions were found in Genlisea, Pinguicula, Aldrovanda, Dionaea, Drosera, Drosophyllum, Nepenthes, and Cephalotus. In Utricularia and Genlisea, enzymatic secretion was independent of stimulation by prey. Byblis and Roridula are usually considered as “proto‐carnivores”, lacking digestive enzymes. However, we found high activity of phosphatases in both species. Thus, they should be classified as true carnivores. We suggest that the inflorescence of Byblis and some Pinguicula species might also be an additional “carnivorous organ”, which can trap a prey, digest it, and finally absorb available nutrients.     

Defense and carnivory: Dual role of bracts inPassiflora foetida

Members of the genusPassiflora are reported to have evolved modifications which kill insects; they have however never been tested for carnivorous syndrome. The flowers ofPassiflora foetida consists of highly reticulate bracts which cover and grow along with the buds and fruits. Removal of bracts from developing bud and fruit resulted in higher predatory damage compared to those where the bracts were intact. These bracts also possess a large number of minute glands which ooze sticky secretion. A variety of tiny insects were found trapped by the secretion of the bracts. The secretion of these glands show high proteases and acid phosphatase activity, two common digestive enzymes found in traps of true carnivorous plants. A high quantity of aminoacids were released from freshly freeze killed ants when incubated in buffer extract of bracts-[¹⁴C] phenylalanine smeared on the glandular surface of bracts was recovered from ovules suggesting potential for absorption of aminoacids. These results suggest a novel role for bracts where primary function is to minimize predatory damage to developing flowers and fruits. The bracts serve as insect traps and also possess the mechanism to digest the trapped insects to obtain free aminoacids.