Plant peptides and peptidomics

Extracellular plant peptides perform a large variety of functions, including signalling and defence. Intracellular peptides often have physiological functions or may merely be the products of general proteolysis. Plant peptides have been identified and, in part, functionally characterized through biochemical and genetic studies, which are lengthy and in some cases impractical. Peptidomics is a branch of proteomics that has been developed over the last 5 years, and has been used mainly to study neuropeptides in animals and the degradome of proteases. Peptidomics is a fast, efficient methodology that can detect minute and transient amounts of peptides and identify their post-translational modifications. This review describes known plant peptides and introduces the use of peptidomics for the detection of novel plant peptides.     

Antimicrobial Peptides from Plants

Peptides with antimicrobial properties are present in most if not all plant species. All plant antimicrobial peptides isolated so far contain even numbers of cysteines (4, 6, or 8), which are all pairwise connected by disulfide bridges, thus providing high stability to the peptides. Based on homologies at the primary structure level, plant antimicrobial peptides can be classified into distinct families including thionins, plant defensins, lipid transfer proteins, and he vein- and knottin-type antimicrobial peptides. Detailed three-dimensional structure information has been obtained for one or more members of these peptide families. All antimicrobial peptides studied thus far appear to exert their antimicrobial effect at the level of the plasma membrane of the target microorganism, but the different peptide types are likely to act via different mechanisms. Antimicrobial peptides can occur in all plant organs. In unstressed organs, antimicrobial peptides are usually most abundant in the outer cell layer lining the organ, which is consistent with a role for the antimicrobial peptides in constitutive host defense against microbial invaders attacking from the outside. Thionins are predominantly located intracellularly but are also found in the extracellular space, whereas most plant defensins and lipid transfer proteins are deposited exclusively in the extracellular space. In a number of plant species, a strong induction of genes expressing either thionins, plant defensins, or lipid transfer proteins has been observed on infection of the leaves by microbial pathogens. Hence, antimicrobial peptides can also take part in the inducible defense response of plants. Constitutive expression in transgenic plants of heterologous antimicrobial peptide genes has been achieved, which in some cases has led to enhanced resistance to particular microbial plant pathogens.     

Role of plant hormones in plant defence responses

Plant hormones play important roles in regulating developmental processes and signaling networks involved in plant responses to a wide range of biotic and abiotic stresses. Significant progress has been made in identifying the key components and understanding the role of salicylic acid (SA), jasmonates (JA) and ethylene (ET) in plant responses to biotic stresses. Recent studies indicate that other hormones such as abscisic acid (ABA), auxin, gibberellic acid (GA), cytokinin (CK), brassinosteroids (BR) and peptide hormones are also implicated in plant defence signaling pathways but their role in plant defence is less well studied. Here, we review recent advances made in understanding the role of these hormones in modulating plant defence responses against various diseases and pests.     

The role of plant defence proteins in fungal pathogenesis

It is becoming increasingly evident that a plant–pathogen interaction may be compared to an open warfare, whose major weapons are proteins synthesized by both organisms. These weapons were gradually developed in what must have been a multimillion-year evolutionary game of ping-pong. The outcome of each battle results in the establishment of resistance or pathogenesis. The plethora of resistance mechanisms exhibited by plants may be grouped into constitutive and inducible, and range from morphological to structural and chemical defences. Most of these mechanisms are defensive, exhibiting a passive role, but some are highly active against pathogens, using as major targets the fungal cell wall, the plasma membrane or intracellular targets. A considerable overlap exists between pathogenesis-related (PR) proteins and antifungal proteins. However, many of the now considered 17 families of PR proteins do not present any known role as antipathogen activity, whereas among the 13 classes of antifungal proteins, most are not PR proteins. Discovery of novel antifungal proteins and peptides continues at a rapid pace. In their long coevolution with plants, phytopathogens have evolved ways to avoid or circumvent the plant defence weaponry. These include protection of fungal structures from plant defence reactions, inhibition of elicitor-induced plant defence responses and suppression of plant defences. A detailed understanding of the molecular events that take place during a plant–pathogen interaction is an essential goal for disease control in the future.     

The effect of C-terminal amidation on the efficacy and selectivity of antimicrobial and anticancer peptides

Cationic defence peptides show high therapeutic potential as antimicrobial and anticancer agents. Some of these peptides carry a C-terminal amide moiety which has been shown to be required for antimicrobial activity. However, whether this is a general requirement or whether C-terminal amidation is required for the anticancer activity of defence peptides is unclear. In response, this study analyses the toxicity of a series of C-terminally amidated defence peptides and their non-amidated isoforms to normal fibroblast cells, a variety of tumour cells and bacterial cells. The toxicities of these peptides to microbial and cancer cells were generally <200 μM. Peptides were either unaffected by C-terminal amidation or showed up to 10-fold decreases or increases in efficacy. However, these peptides all showed toxicity to normal fibroblast cells with levels (generally <150 μM) that were comparable to those of their antimicrobial and anticancer activities. In contrast to previous claims which have been based on analysis of single amidation events, the results of this study clearly show that the C-terminal amidation of defence peptides has a variable effect on their antimicrobial and anticancer efficacy and no clear effect on their selectivity for these cell types.     

Cooperative interaction of antimicrobial peptides with the interrelated immune pathways in plants

Plants express a diverse repertoire of functionally and structurally distinct antimicrobial peptides (AMPs) which provide innate immunity by acting directly against a wide range of pathogens. AMPs are expressed in nearly all plant organs, either constitutively or in response to microbial infections. In addition to their direct activity, they also contribute to plant immunity by modulating defence responses resulting from pathogen‐associated molecular pattern/effector‐triggered immunity, and also interact with other AMPs and pathways involving mitogen‐activated protein kinases, reactive oxygen species, hormonal cross‐talk and sugar signalling. Such links among AMPs and defence signalling pathways are poorly understood and there is no clear model for their interactions. This article provides a critical review of the empirical data to shed light on the wider role of AMPs in the robust and resource‐effective defence responses of plants.     

On the study of plant defence and herbivory using comparative approaches: how important are secondary plant compounds

Species comparisons are a cornerstone of biology and there is a long tradition of using the comparative framework to study the ecology and evolution of plant defensive traits. Early comparative studies led to the hypothesis that plant chemistry plays a central role in plant defence, and the evolution of plant secondary chemistry in response to insect herbivory remains a classic example of coevolution. However, recent comparative work has disagreed with this paradigm, reporting little connection between plant secondary chemicals and herbivory across distantly related plant taxa. One conclusion of this new work is that the importance of secondary chemistry in plant defence may have been generally overstated in earlier research. Here, we attempt to reconcile these contradicting viewpoints on the role of plant chemistry in defence by critically evaluating the use and interpretation of species correlations as a means to study defence–herbivory relationships. We conclude that the notion that plant primary metabolites (e.g. leaf nitrogen content) are the principal determinants of herbivory (or the target of natural selection by herbivores) is not likely to be correct. Despite the inference of recent community‐wide studies of herbivory, strong evidence remains for a prime role of secondary compounds in plant defence against herbivores.     

Optimal defence, kin conflict and the distribution of furanocoumarins among offspring of wild parsnip

The factors influencing the allocation of chemical defences to plant offspring have largely been unexplored, conceptually and experimentally. Because evolutionary interactions between maternal plants and their progeny can affect resource allocation patterns among sibling offspring, we suggest that kin conflict as well as herbivore–plant interaction theories need to be considered to predict chemical defence allocation patterns. Optimal defence theory predicts that maternal plants should defend more heavily those offspring in which resources have been disproportionately invested. In contrast, kin conflict theory predicts that natural selection will favour genotypes that can compete successfully for maternal defences irrespective of their quality, even at the expense of the fitness of siblings and the maternal plant. Evidence for these defence patterns were evaluated by examining the allocation of furanocoumarins to seeds of the wild parsnip (Pastinaca sativa, Apiaceae). Furanocoumarins are toxins that are localized within the oil tubes of the maternal tissues of seeds. We evaluated the role of offspring investment (endosperm mass) and seed genotype on furanocoumarin allocation by mating an array of pollen donors with pollen recipients. Furanocoumarins were found to be positively correlated with endosperm mass on one side of the seed, a result consistent with optimal defence theory; however, on the other side of the seed, furanocoumarin content was influenced by seed genotype and was unrelated to endosperm mass. These effects varied with maternal plant. Further experiments demonstrated that nearly 80% of furanocoumarin production occurs after pollination, when fertilization products are active. Although the amount of furanocoumarin influenced by the seed genotype is small relative to the total quantity in the seed, these furanocoumarins are the first line of defence against important predators, such as the parsnip webworm, Depressaria pastinacella (Lepidoptera: Oecophoridae). We found that parsnip webworm larvae were able to discriminate among genotypes within an inflorescence. In line with previous studies, these results suggest that a genotype's ability to influence furanocoumarin defence may affect its probability of survival. We conclude that the distribution of defences among plant offspring in wild parsnip is probably influenced by competition among seed genotypes that conflicts with maternal optimal defence. This revised version was published online in July 2006 with corrections to the Cover Date.     

Plant signalling peptides

Biochemical and genetic studies have identified peptides that play crucial roles in plant growth and development, including defence mechanisms in response to wounding by pests, the control of cell division and expansion, and pollen self-incompatibility. The first two signalling peptides to be described in plants were tomato systemin and phytosulfokine (PSK). There is also biochemical evidence that natriuretic peptide-like molecules, immunologically-related to those found in animals, may exist in plants. Another example of signalling peptide is ENOD40, a product of a gene, which became active early in the root nodulation process following Rhizobium infection of legumes. Other predicted bioactive peptides or oligopeptides have been identified by means of genetic, rather then biochemical methods. The Arabidopsis CLAVATA3 protein is required for the correct organization of the shoot apical meristem and the pollen S determinant S-locus cysteine-rich protein (SCR) also called S-locus protein 11, SP11). The plant signalling peptides discovered so far are involved in various processes and play an important role in communication between cells or organs, respectively. This review will focus on these peptides and their role in intercellular signalling.     

Salicylic acid has a dual role in the activation of defence-related genes in parsley

Systemic acquired resistance is an inducible plant defence state, the activation of which depends mostly on the accumulation of salicylic acid (SA). During the past several years, it has been demonstrated that pretreatment of cultured parsley cells with SA potentiates the elicitation of several defence responses that are local in whole plants, including the accumulation of phenylpropanoid products. Here it is reported that while anionic peroxidase and mannitol dehydrogenase encoding genes are directly responsive to SA, pretreating parsley cells with SA not only enhances elicitation of the phenylpropanoid genes phenylalanine ammonia-lyase and 4-coumarate:CoA ligase but also of genes for PR-10 and a hydroxyproline-rich glycoprotein. Enhanced induction of these genes was seen at low levels of endogenous free SA. Enhancement of phenylalanine ammonia-lyase gene activation was proportional to the length of SA pretreatment. Furthermore, the ability of SA analogues to both potentiate elicited and directly induce defence gene activation correlated with their biological activity to promote plant disease resistance. In summary, these results emphasize that SA has at least a dual role in plant defence gene activation.     

Switching on plant genes by external chemical signals

During the past decade there has been rapidly increasing interest in the role of plant volatiles in insect-plant interactions and the induction of plant defence systems by both pathogens and herbivores. Scientists are striving to link the proximate studies elucidating pathways and genes with the ultimate adaptive studies that attempt to explain their ecological role. However, we still do not know whether plants 'talk' to one another by employing 'phytopheromones'.     

Exopolysaccharides produced by plant pathogenic bacteria affect ascorbate metabolism in Nicotiana tabacum

The role of the exopolysaccharides (EPSs) produced by plant pathogenic bacteria has not completely clarified, they are considered either molecules able to avoid or delay the activation of plant defences, or acting as signal in the plant-pathogen cross-talk. In order to understand whether EPSs are recognized by infected plant cells and are able to induce the activation of plant defence responses, their capability to induce metabolic alteration in tobacco cells has been analysed. The results indicate that several EPSs, even if not chemically related, induce increases in phenylalanine ammonia-lyase, a marker enzyme of defence responses of plants against stress; but others are completely ineffective. The EPSs affecting phenylalanine ammonia-lyase also induce an increase in hydrogen peroxide production. Moreover, they alter the metabolism of ascorbate, another parameter indicative of the presence of stress conditions and the involvement of which in the hypersensitive reaction has been recently reported. The possibility that specific EPSs could act as signals in the plant-pathogenic bacteria interaction is discussed.     

Polyamines and plant disease

The diamine putrescine and the polyamines spermidine and spermine are found in a wide range of organisms from bacteria to plants and animals. They are basic, small molecules implicated in the promotion of plant growth and development by activating the synthesis of nucleic acids. Polyamine metabolism has long been known to be altered in plants responding to abiotic environmental stress and to undergo profound changes in plants interacting with fungal and viral pathogens. Polyamines conjugated to phenolic compounds, hydroxycinnamic acid amides (HCAAs), have been shown to accumulate in incompatible interactions between plants and a variety of pathogens, while changes in the diamine catabolic enzyme diamine oxidase suggest a role for this enzyme in the production of hydrogen peroxide during plant defence responses. More recent work has suggested a role for the free polyamine spermine in the hypersensitive response of barley to powdery mildew and particularly in tobacco to TMV. The prospects for the genetic manipulation of HCAA levels in plants as a means of both defining their role in plant defence and in the generation of disease resistant plants is discussed briefly.     

Plant-mediated effects in the Brassicaceae on the performance and behaviour of parasitoids

Direct and indirect plant defences are well studied, particularly in the Brassicaceae. Glucosinolates (GS) are secondary plant compounds characteristic in this plant family. They play an important role in defence against herbivores and pathogens. Insect herbivores that are specialists on brassicaceous plant species have evolved adaptations to excrete or detoxify GS. Other insect herbivores may even sequester GS and employ them as defence against their own antagonists, such as predators. Moreover, high levels of GS in the food plants of non-sequestering herbivores can negatively affect the growth and survival of their parasitoids. In addition to allelochemicals, plants produce volatile chemicals when damaged by herbivores. These herbivore induced plant volatiles (HIPV) have been demonstrated to play an important role in foraging behaviour of insect parasitoids. In addition, biosynthetic pathways involved in the production of HIPV are being unraveled using the model plant Arabidopsis thialiana. However, the majority of studies investigating the attractiveness of HIPV to parasitoids are based on experiments mainly using crop plant species in which defence traits may have changed through artificial selection. Field studies with both cultivated and wild crucifers, the latter in which defence traits are intact, are necessary to reveal the relative importance of direct and indirect plant defence strategies on parasitoid and plant fitness. Future research should also consider the potential conflict between direct and indirect plant defences when studying the evolution of plant defences against insect herbivory.     

Anionic Antimicrobial and Anticancer Peptides from Plants

Anionic antimicrobial peptides (AAMPs) have been identified in a wide variety of plant species with net charges that range between ?1 and ?7 and structures that include: extended conformations, α-helical architecture and cysteine stabilized scaffolds. These peptides commonly exist as multiple isoforms within a given plant and have a range of biological activities including the ability to kill cancer cells as well as phytopathogenic bacteria, fungi, pests, molluscs, and other predatory species. In general, the killing mechanisms underpinning these activities are poorly understood although they appear to involve attack on intracellular targets such as DNA along with compromise of cell envelope integrity through lysis of the cell wall via chitin-binding and/or permeabilisation of the plasma membrane via lipid interaction. It is now becoming clear that AAMPs participate in the innate immune response of plants and make a major contribution to the arsenal of defence toxins produced by these organisms to compensate for their lack of some defence mechanisms possessed by mammals, such as mobility and a somatic adaptive immune system. Based on their biological properties, a number of potential uses for plant AAMPs have been suggested, including therapeutically useful anticancer agents and novel antimicrobial compounds, which could be utilized in a variety of scenarios, ranging from the protection of crops to the disinfection of hospital environments.     

Proteases in pathogenesis and plant defence

Plant pathogens deliver a variety of virulence factors to host cells to suppress basal defence responses and create suitable environments for their propagation. Plants have in turn evolved disease resistance genes whose products detect the virulence factors as a signal of invasion and activate effective defence responses. Understanding how a virulence effector contributes to virulence on susceptible hosts but becomes an avirulence factor that triggers defence responses on resistance hosts has been a major focus in plant research. Recent studies have shown that a growing list of pathogen-encoded effectors functions as proteases that are secreted into plant cells to modify host proteins. In addition, several plant proteases have been found to function in activation of the defence mechanism. These findings reveal that post-translational modification of host proteins through proteolytic processing is a widely used mechanism in regulating the plant defence response.     

Rethinking the role of many plant phenolics – protection from photodamage not herbivores?

Phenolics have been considered classic defence compounds for protecting plants from herbivores, ever since plant secondary metabolites were suggested to have evolved for that reason. The resource availability and carbon-nutrient balance hypotheses proposed that variation in phenolic levels between and within plant species reflects environmental availability of nutrients and light, and represents a trade-off in allocation by plants to growth and defence against herbivores. In contrast to these concepts, we suggest that (1) the main role of many plant phenolics may be to protect leaves from photodamage, not herbivores; (2) they can achieve this by acting as antioxidants; and (3) their levels may vary with environmental conditions in order to counteract this potential photodamage. We therefore suggest that patterns in phenolic levels, often used to support the concept of trade-off between growth and herbivore defence in relation to resource availability, may actually reflect different risks of photodamage. We suggest that the level of many phenolics is low under some environmental conditions, not because resources to produce them are limited, but simply because the risk of photodamage is low and they are not required. If our photodamage hypothesis is correct, a reassessment of the ecological and evolutionary role of many phenolics in plant defence theory is required.     

In the trenches of plant pathogen recognition: Role of NB-LRR proteins

As in nearly every discipline of plant biology, new insights are constantly changing our understanding of plant immunity. It is now clear that plant immunity is controlled by two layers of inducible responses: basal responses triggered by conserved microbial features and specific responses triggered by gene-for-gene recognition of pathogen effector proteins by host resistance (R) proteins. The nucleotide-binding domain leucine-rich repeat (NB-LRR) class of R proteins plays a major role in the combat against a wide range of plant pathogens. The variation that has been generated and is maintained within these conserved proteins has diversified their specificity, subcellular localisations, activation and recognition mechanisms, allowing them to specifically adapt to different plant–pathogen interaction systems. This review addresses recent advances in the molecular role of NB-LRR proteins in pathogen recognition and activation of plant defence responses.     

The increasing role of phosphatidylethanolamine as a lipid receptor in the action of host defence peptides

Host defence peptides (HDPs) are antimicrobial agents produced by organisms across the prokaryotic and eukaryotic kingdoms. Many prokaryotes produce HDPs, which utilise lipid and protein receptors in the membranes of bacterial competitors to facilitate their antibacterial action and thereby survive in their niche environment. As a major example, it is well established that cinnamycin and duramycins from Streptomyces have a high affinity for phosphatidylethanolamine (PE) and exhibit activity against other Gram-positive organisms, such as Bacillus. In contrast, although eukaryotic HDPs utilise membrane interactive mechanisms to facilitate their antimicrobial activity, the prevailing view has long been that these mechanisms do not involve membrane receptors. However, this view has been recently challenged by reports that a number of eukaryotic HDPs such as plant cyclotides also use PE as a receptor to promote their antimicrobial activities. Here, we review current understanding of the mechanisms that underpin the use of PE as a receptor in the antimicrobial and other biological actions of HDPs and describe medical and biotechnical uses of these peptides, which range from tumour imaging and detection to inclusion in topical microbicidal gels to prevent the sexual transmission of HIV.