A role for plant natriuretic peptide immuno-analogues in NaCl- and drought-stress responses

Higher plants contain biologically active molecules that are recognized by anti-human atrial natriuretic polypeptide rabbit serum (anti-ANP). These molecules are termed immunoreactant plant natriuretic peptides (irPNPs) and have previously been shown to be associated with conductive tissue and to affect ion fluxes, protoplast volume regulation and stomatal guard cell responses. Herein an irPNP from the brassicaceus weed Erucastrum strigosum is identified and it is demonstrated that the relative amounts of irPNP expressed as a percentage of total water : methanol (50 : 50) extracted proteins are increased when plants are exposed to 300 m M NaCl. Since 100 and 200 m M NaCl reduce dry and fresh mass as well as increase total tissue NaCl load, it is hypothesized that irPNP up-regulation is a late and possibly adaptive response. IrPNP is also significantly up-regulated in Arabidopsis thaliana suspension culture cells in response to 150 m M NaCl and even more so in response to iso-osmolar amounts of sorbitol. Finally, a recombinant A. thaliana irPNP (AtPNP-A) promotes net water-uptake into the protoplast and thus volume increases. This response is dependent on de novo protein synthesis and may suggest a complex and possibly regulatory function for irPNP-like molecules in plant homeostasis.     

Plant natriuretic peptides are apoplastic and paracrine stress response molecules

Higher plants contain biologically active proteins that are recognized by antibodies against human atrial natriuretic peptide (ANP). We identified and isolated two Arabidopsis thaliana immunoreactive plant natriuretic peptide (PNP)-encoding genes, AtPNP-A and AtPNP-B, which are distantly related members of the expansin superfamily and have a role in the regulation of homeostasis in abiotic and biotic stresses, and have shown that AtPNP-A modulates the effects of ABA on stomata. Arabidopsis PNP (PNP-A) is mainly expressed in leaf mesophyll cells, and in protoplast assays we demonstrate that it is secreted using AtPNP-A:green fluorescent protein (GFP) reporter constructs and flow cytometry. Transient reporter assays provide evidence that AtPNP-A expression is enhanced by heat, osmotica and salt, and that AtPNP-A itself can enhance its own expression, thereby generating a response signature diagnostic for paracrine action and potentially also autocrine effects. Expression of native AtPNP-A is enhanced by osmotica and transiently by salt. Although AtPNP-A expression is induced by salt and osmotica, ABA does not significantly modulate AtPNP-A levels nor does recombinant AtPNP-A affect reporter expression of the ABA-responsive RD29A gene. Together, these results provide experimental evidence that AtPNP-A is stress responsive, secreted into the apoplastic space and can enhance its own expression. Furthermore, our findings support the idea that AtPNP-A, together with ABA, is an important component in complex plant stress responses and that, much like in animals, peptide signaling molecules can create diverse and modular signals essential for growth, development and defense under rapidly changing environmental conditions.     

AtPNP-A is a systemically mobile natriuretic peptide immunoanalogue with a role in Arabidopsis thaliana cell volume regulation

Cellular and physiological evidence suggests the presence of a novel class of systemically mobile plant molecules that are recognized by antibodies against vertebrate atrial natriuretic peptides (ANPs). In order to characterize the function of these immunoanalogues we have expressed the full-length recombinant (AtPNP-A[1-126]) and demonstrate that this molecule induces osmoticum-dependent H(2)O uptake into protoplasts at nanomolar concentrations and thus affects cell volume. A similar response is also seen with a recombinant that does not contain the signal peptide (AtPNP-A[26-126]) as well as a short domain (AtPNP-A[33-66]) that shows homology to the vertebrate peptide. Taken together, these findings suggest that AtPNP-A has an important and systemic role in plant growth and homeostasis.     

A recombinant plant natriuretic peptide causes rapid and spatially differentiated K+, Na+ and H+ flux changes in Arabidopsis thaliana roots

Plant natriuretic peptides (PNPs) belong to a novel class of systemically mobile molecules that are structurally similar to the N-terminal domain of expansins and affect physiological processes such as protoplast volume regulation at nano-molar concentrations. Here we demonstrate that AtPNP-A, a recombinant Arabidopsis thaliana PNP causes rapid H(+) influx in the elongation zone of A. thaliana roots but not in the mature zone. AtPNP-A also induces significant K(+) and Na(+) efflux and this effect is seen in the mature root zone only. These observations suggest that responses to AtPNP-A are developmental stage and tissue specific and point to a complex role in plant growth and homeostasis.     

The Arabidopsis thaliana natriuretic peptide AtPNP-A is a systemic regulator of leaf dark respiration and signals via the phloem

Plant natriuretic peptides (PNPs) belong to a novel class of peptidic signaling molecules that share some structural similarity to the N-terminal domain of expansins and affect physiological processes such as water and ion homeostasis at nano-molar concentrations. Here we show that a recombinant Arabidopsis thaliana PNP (AtPNP-A) rapidly increased the rate of dark respiration in treated leaves after 5 min. In addition, we observed increases in lower leaves, and with a lag time of 10 min, the effect spread to the upper leaves and subsequently (after 15 min) to the opposite leaves. This response signature is indicative of phloem mobility of the signal, a hypothesis that was further strengthened by the fact that cold girdling, which affects phloem but not xylem or apoplastic processes, delayed the long distance AtPNP-A effect. We conclude that locally applied AtPNP-A can induce a phloem-mobile signal that rapidly modifies plant homeostasis in distal parts.     

In situ localization associates biologically active plant natriuretic peptide immuno-analogues with conductive tissue and stomata

Plant natriuretic peptide immuno-analogues (irPNP) have previously been shown to affect a number of biological processes including stomatal guard cell movements, ion fluxes and osmoticum-dependent water transport. Tissue printing and immunofluorescent labelling techniques have been used here to study the tissue and cellular localization of irPNP in ivy (Hedera helix L.) and potato (Solanum tuberosum L.). Polyclonal antibodies active against human atrial natriuretic peptide (anti-hANP) and antibodies against irPNP from potato (anti-StPNP) were used for immunolabelling. Tissue prints revealed that immunoreactants are concentrated in vascular tissues of leaves, petioles and stems. Phloem-associated cells, xylem cells and parenchymatic xylem cells showed the strongest immunoreaction. Immunofluorescent microscopy with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG supported this finding and, furthermore, revealed strong labelling to stomatal guard cells and the adjacent apoplastic space as well. Biologically active immunoreactants were also detected in xylem exudates of a soft South African perennial forest sage (Plectranthus ciliatus E. Mey ex Benth.) thus strengthening the evidence for a systemic role of the protein. In summary, in situ cellular localization is consistent with physiological responses elicited by irPNPs reported previously and is indicative of a systemic role in plant homeostasis.     

Co-expression and promoter content analyses assign a role in biotic and abiotic stress responses to plant natriuretic peptides

Background  

Plant natriuretic peptides (PNPs) are a class of systemically mobile molecules distantly related to expansins. While several physiological responses to PNPs have been reported, their biological role has remained elusive. Here we use a combination of expression correlation analysis, meta-analysis of gene expression profiles in response to specific stimuli and in selected mutants, and promoter content analysis to infer the biological role of the Arabidopsis thaliana PNP, AtPNP-A.     

Lipo-Chitin Oligosaccharides,Plant Symbiosis Signalling Molecules That Modulate Mammalian Angiogenesis In Vitro

Lipochitin oligosaccharides (LCOs) are signaling molecules required by ecologically and agronomically important bacteria and fungi to establish symbioses with diverse land plants. In plants, oligo-chitins and LCOs can differentially interact with different lysin motif (LysM) receptors and affect innate immunity responses or symbiosis-related pathways. In animals, oligo-chitins also induce innate immunity and other physiological responses but LCO recognition has not been demonstrated. Here LCO and LCO-like compounds are shown to be biologically active in mammals in a structure dependent way through the modulation of angiogenesis, a tightly-regulated process involving the induction and growth of new blood vessels from existing vessels. The testing of 24 LCO, LCO-like or oligo-chitin compounds resulted in structure-dependent effects on angiogenesis in vitro leading to promotion, or inhibition or nil effects. Like plants, the mammalian LCO biological activity depended upon the presence and type of terminal substitutions. Un-substituted oligo-chitins of similar chain lengths were unable to modulate angiogenesis indicating that mammalian cells, like plant cells, can distinguish between LCOs and un-substituted oligo-chitins. The cellular mode-of-action of the biologically active LCOs in mammals was determined. The stimulation or inhibition of endothelial cell adhesion to vitronectin or fibronectin correlated with their pro- or anti-angiogenic activity. Importantly, novel and more easily synthesised LCO-like disaccharide molecules were also biologically active and de-acetylated chitobiose was shown to be the primary structural basis of recognition. Given this, simpler chitin disaccharides derivatives based on the structure of biologically active LCOs were synthesised and purified and these showed biological activity in mammalian cells. Since important chronic disease states are linked to either insufficient or excessive angiogenesis, LCO and LCO-like molecules may have the potential to be a new, carbohydrate-based class of therapeutics for modulating angiogenesis.     

Strigolactones: New Physiological Roles for an Ancient Signal

Strigolactones are an ancient group of plant signalling molecules. They play a critical role in the rhizosphere where they facilitate the formation of symbioses with fungi, crucial for the acquisition of plant nutrients in over 80 % of land plant species. Strigolactones have also been exploited by parasitic weeds as a rhizosphere signal indicating the presence of a host species, resulting in devastating losses in some agricultural systems. Recently, they have also been shown to act endogenously as plant hormones controlling shoot branching and have been implicated in a wide range of other physiological processes, including root growth, root-hair elongation, adventitious rooting, secondary growth, photomorphogenesis, seed germination, nodulation, and protonemal development in mosses. Here, we discuss the evidence for the involvement of strigolactones as endogenous regulators of these processes and highlight some examples where the evidence is inconclusive. One major gap in our understanding is the identity of the endogenous strigolactone(s) that are biologically active. A discussion of the interactions between the different plant hormones and the possible role of strigolactones as integrators of the root-to-shoot balance, nutrient acquisition, and thus resource allocation illustrates some important future directions for this area of research.     

A new method of producing biologically active nanocomplexes by non-covalent conjugation of proteins with viral particles

Currently, a range of biologically active molecules have been attached to plant and bacterial viras nanoscaffolds, yielding stable nanoparticles that display multiple copies of the desired molecule. In this paper we propose a new method of non-covalent attachment of peptides to the surface of virios. We have demonstrated that this method is efficient in a model system that includes tobacco mosaic virus particles, synthetic polycation (quaternized poly(4-vinylpyridine) carrying ethyl ethyl pendant radicals) and polypeptide of interest. This principle of step-by-step binding to the surface of virions was used for electrostatic association with hydrophilic fragment of influenza virus haemagglutinin.     

Structural identification of a novel pro-inflammatory epoxyisoprostane phospholipid in mildly oxidized low density lipoprotein.

One of the earliest steps in the development of the atherosclerotic lesion is the accumulation of monocyte/macrophages within the vessel wall. Oxidized lipids present in minimally modified-low density lipoproteins (MM-LDL) contribute to this process by activating endothelial cells to express monocyte-specific adhesion molecules and chemoattractant factors. A major focus of our group has been the isolation and characterization of the biologically active oxidized lipids in MM-LDL. We have previously characterized three oxidized phospholipids present in MM-LDL, atherosclerotic lesions of fat fed rabbits, and autoxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) that induced human aortic endothelial cells to adhere human monocytes in vitro. We have used sequential normal and reverse phase-high performance liquid chromatography to isolate various isomers of an oxidized phospholipid from autoxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine. The fatty acid in the sn-2 position of this biologically active isomer and its dehydration product was released by phospholipase A(2) and characterized. Hydrogenation with platinum(IV) oxide/hydrogen suggested a cyclic moiety, and reduction with sodium borohydride suggested two reducible oxygen-containing groups in the molecule. The fragmentation pattern produced by electrospray ionization-collision induced dissociation-tandem mass spectrometry was consistent with a molecule resembling an E-ring prostaglandin with an epoxide at the 5,6 position. The structure of this lipid was confirmed by proton nuclear magnetic resonance spectroscopy analysis of the free fatty acid isolated from the dehydration product of m/z 828.5. Based on these studies, we arrived at the structure of the biologically active oxidized phospholipids as 1-palmitoyl-2-(5, 6-epoxyisoprostane E(2))-sn-glycero-3-phosphocholine. The identification of this molecule adds epoxyisoprostanes to the growing list of biologically active isoprostanes.     

Inhibition of caspase-1 in rat brain reduces spontaneous nonrapid eye movement sleep and nonrapid eye movement sleep enhancement induced by lipopolysaccharide

Evidence suggests that IL-1beta is involved in promoting physiological nonrapid eye movement (NREM) sleep. IL-1beta has also been proposed to mediate NREM sleep enhancement induced by bacteria or their components. Mature and biologically active IL-1beta is cleaved from an inactive precursor by a cysteinyl aspartate-specific protease (caspase)-1. This study aimed to test the hypothesis that inhibition in brain of the cleavage of biologically active IL-1beta will reduce in rats both spontaneous NREM sleep and NREM sleep enhancement induced by the peripheral administration of components of the bacterial cell wall. To test this hypothesis, rats were intracerebroventricularly administered the caspase-1 inhibitor Ac-Tyr-Val-Ala-Asp chloromethyl ketone (YVAD; 3, 30, 300, and 1,500 ng) or were pretreated intracerebroventricularly with YVAD (300 ng) and then intraperitoneally injected with the gram-negative bacterial cell wall component LPS (250 microg/kg). Subsequent sleep-wake behavior was determined by standard polygraphic recordings. YVAD administration at the beginning of the light phase of the light-dark cycle significantly reduced time spontaneously spent in NREM sleep during the first 12 postinjection hours. YVAD pretreatment also completely prevented NREM sleep enhancement induced by peripheral LPS administration at the beginning of the dark phase. These results, in agreement with previous evidence, support the involvement of brain IL-1beta in physiological promotion of NREM sleep and in mediating NREM sleep enhancement induced by peripheral immune challenge.     

Small molecules: the lexicon of biodiversity

The extent of microbial diversity in the Biosphere is not known (and probably never will be!). One aspect of this diversity is the production of biologically active small molecules; within the Streptomycetes alone this may be millions of compounds with an extraordinary diversity and complexity of structure. First recognised as pigments and later, in the 1950s, as antibiotics, it is now clear that the small molecules produced by microbes have many different functions in nature. This huge collection of biologically active compounds with various properties has been used as pharmaceuticals and agriculturals. They also interact with proteins and RNA with high specificity and have been shown to be regulators and effectors of diverse biochemical reactions. The use of small molecules (other than as pharmaceuticals) deserves to be explored in order to exploit microbial biotechnology to the full.     

Recent developments in the use of transgenic plants for the production of human therapeutics and biopharmaceuticals

In recent years there has been a dramatic increase in the application of plant biotechnology for the production of a variety of commercially valuable simple and complex biological molecules (biologics) for use in human and animal healthcare. Transgenic whole plants and plant cell culture systems have been developed that have the capacity to economically produce large-scale quantities of antibodies and antibody fragments, antigens and/or vaccine epitopes, metabolic enzymes, hormones, (neuro)peptides and a variety of biologically active complexes and secondary metabolites for direct use as therapeutic agents or diagnostic tools in the medical healthcare industry. As the products of genetically modified plants make their way from concept to commercialization the associated risks and acceptance by the public has been become a focal point. In this paper, we summarize the recent advances made in the use of transgenic plants and plant cell cultures as biological factories for the production of human therapeutics and biopharmaceuticals and discuss the long-term potential of `molecular farming' as a low-cost, efficient method for the production of biological materials with demonstrated utility to the pharmaceutical industry or medical community.     

Nanodiamond bullets and their biological targets

The potential of nanodiamond bullets for use in ballistic delivery of biologically active molecules was investigated. Detonation nanodiamond particles were coated with DNA, synthetic ethylene precursor, or ethylene antagonist or were labeled with fluorescent dyes and delivered into bacteria, yeast, insect cells, and plant tissues with the help of a Bio-Rad particle delivery system PDS-1000/He. Detonation nanodiamonds are both mechanically and chemically stable and can be used as DNA carriers for biolistic transformation of cells and in delivery of biologically active molecules.     

Novel Thionins from Black Seed (<Emphasis Type="Italic">Nigella sativa</Emphasis> L.) Demonstrate Antimicrobial Activity

Black seed (Nigella sativa) is known as a good source of various biologically active compounds which possess antimicrobial properties. One of our objectives was to elaborate methods of obtaining and extracting peptides from plants. In the current study, we discovered some biological effects of thionins from black seed, such as bactericidal and fungicidal effects. Isolation of thionins performed by combining acidic extraction and fractionation with various liquid chromatography methods. The N-terminal amino acid sequences were revealed using automated Edman degradation. The antimicrobial activity of thionins were evaluated by a microdilution broth assay. A fluorescent spectroscopy and an atomic force microscopy allow to investigate the features of mode of action of the thionins. The two novel peptides from black seed (N. sativa L.), a plant endemic to Central Asia. These peptides, named NsW1 and NsW2, have a high affinity with heparin, a polysaccharide glycosaminoglycan. These molecules were indentified as thionins, a well-known family of plant antimicrobial peptides. These thionins effectively inhibit viability of Bacillus subtilus, Staphylococcus aureus and Candida albicans that has been confirmed using a bacteriological and some biophysical techniques. Obtained data indicate that black seed thionins are biologically active molecules that may be considered to be perspective antibacterial agents.     

Mating type-specific cell-cell recognition of Saccharomyces cerevisiae: cell wall attachment and active sites of a- and alpha-agglutinin.

Mating type-specific agglutination of Saccharomyces cerevisiae a and alpha cells depends on the heterophilic interaction of two cell surface glycoproteins, the gene products of AG alpha 1 and AGA2. Evidence is presented with immunogold labelling that the alpha-agglutinin is part of the outer fimbrial cell wall coat. The a-agglutinin is bound via two S-S bridges (Cys7 and Cys50) to a cell wall component, most probably the gene product of AGA1. His273 of alpha-agglutinin has previously been shown to be essential for a- and alpha-agglutinin interaction and a model based on two opposing ion-pairs had been proposed. By site-directed mutagenesis this possibility has now been excluded. With the help of various peptides, either chemically synthesized, obtained by proteolysis of intact glycosylated a-agglutinin or prepared from a fusion protein expressed in Escherichia coli, the biologically active region of a-agglutinin was located at the C-terminus of the molecule. A peptide consisting of the C-terminal 10 amino acids (GSPIN-TQYVF) was active in nanomolar concentrations. Saccharide moieties, therefore, are not essential for the mating type-specific cell-cell interaction; glycosylated peptides are, however, four to five times more active than non-glycosylated ones. Comparisons of the recognition sequences of the S. cerevisiae agglutinins with that of the Dictyostelium contact site A glycoprotein (gp80), as well as with those of the various families of cell adhesion molecules of higher eucaryotes, have been made and are discussed.     

Green light for polyphosphoinositide signals in plants

Plant genomes lack homologues of the inositol 1,4,5-trisphosphate receptor and protein kinase C, which are important components of the canonical phospholipase C signalling system in animals. Instead, plants seem to utilize alternative downstream signalling molecules, that is, InsP(6) and phosphatidic acid. Inositol lipids may also function as second messengers themselves. By reversible phosphorylation of the inositol headgroup, five biologically active plant polyphosphoinositides can be detected. Protein targets interact with specific polyphosphoinositide isomers via selective lipid-binding domains, thereby altering their intracellular localization and/or enzymatic activity. Such lipid-binding domains have also been used to create GFP based-lipid biosensors to visualize PPIs dynamics in vivo. Here, we highlight some recent advances and ideas on PPIs' role in plant signalling.     

A fruit-specific and developmentally regulated endopolygalacturonase gene from strawberry (Fragaria x ananassa cv. Chandler)

A fruit-specific and developmentally regulated polygalacturonase gene (spG gene) from strawberry (Fragaria x ananassa cv. Chandler) has been cloned and characterized at a molecular and physiological level. Comparison analysis of the corresponding deduced sPG protein have shown that this strawberry gene is similar to Clade A endopolygalacturonase genes. Moreover, the spatio-temporal and hormonal gene expression pattern suggests a close relationship between the expression of this gene and the onset of the strawberry fruit ripening process and agrees with that of the production of oligosaccharins which have already been described as active molecules involved in fruit ripening. The results are discussed in terms of a putative role of this enzyme in the release of oligosaccharins from the strawberry fruit cell wall.