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Global proteomic analysis of Chelidonium majus and Corydalis cava (Papaveraceae) extracts revealed similar defense-related protein compositions
Abstract
Chelidonium majus and Corydalis cava are phylogenetically closely related (Papaveraceae family). The medicinal and pharmaceutical interest in these plants is based on their synthesis of pharmaceutically important compounds, such as alkaloids, flavonoids, phenolic acids and proteins. Ch. majus shoot and C. cava tuber extracts have been used in traditional folk medicine to treat many diseases, such as fungal, bacterial and viral infections, liver disorders, fever, post-traumatic, colic, abdominal and menstrual pains and even cancer. This study attempts to perform a global comparative proteomic analysis of pharmacologically important extracts from these two closely related unsequenced plant species to gain insights into the protein basis of these plant organs and to compare their common and specific proteomic compositions.We used a shotgun proteomic approach combined with label-free protein quantitation according to the exponentially modified protein abundance index (emPAI). In total, a mean number of 228 protein identification results were recorded in C. cava tuber extracts and about 1240 in Ch. majus shoot extracts. Comparative analysis revealed a similar stress and defense-related protein composition of pharmacologically active plant species and showed the presence of different pathogenesis-related and low molecular inducible antimicrobial peptides. These findings could form the basis for further elucidation of the mechanism of the strong pharmacological activities of these medicinal plant extracts.
... Extracts and the milky sap of Greater Celandine have been used in traditional folk medicine to treat papillae, warts, condylomas, which are visible effects of human papilloma virus (HPV) infections. It has been shown that C. majus extracts have antiviral, antitumor, cytotoxic, anti-inflammatory, antifungal, and antibacterial properties [15,16]. Recent findings using 2-D electrophoresis and tandem mass spectrometry have demonstrated that the milky sap of this plant contains at least 20 defense-related proteins [15]. ...
... Moreover, using shotgun proteomic approach combined with label-free protein quantitation we have recently shown that the C. majus whole plant extract contains a diversity of plant pathogenesis-related proteins. This confirmed that the curative effect of plant medicinal preparations could also arise from the concerted action of many low-molecular defense-related proteins with other compounds that could possibly enhance their synergic effect of biological action [16]. One of the relatively abundant proteins identified was nonspecific lipid transfer protein with potential antibacterial activity [16]. ...
... This confirmed that the curative effect of plant medicinal preparations could also arise from the concerted action of many low-molecular defense-related proteins with other compounds that could possibly enhance their synergic effect of biological action [16]. One of the relatively abundant proteins identified was nonspecific lipid transfer protein with potential antibacterial activity [16]. Therefore the aim of the present study was to isolate and characterize nsLTP protein as important functional component of C. majus latex in terms of its antibacterial activity. ...
Results:
obtained using PCR approach with degenerate primers showed nsLTP protein from C. majus root latex, named CmLTP 9.5. The protein consists of 93 aa with a molecular weight of 9.5kDa (NCBI GenBank accession no. KP733898). The mature form of CmLTP 9.5 has a molecular weight of 7.147kDa and the presence of typical eight strictly conserved cysteine residues. A 3D model of CmLTP 9.5 displays a hydrophobic cavity. The isolated protein fraction tested using diffusion method and critical dilution assay showed strong antibacterial activity towards Gram negative Campylobacter jejuni as well as Gram positive Listeria greyi and Clostridium perfringens. Further studies using protein expression system are required to fully understand CmLTP 9.5 mode of action.
... They have been classified into 17 families with different defensive roles [20,21]. Recent findings have shown that PR proteins could be identified in the latex of latex-bearing plant species [22][23][24][25][26][27][28][29][30][31] and could play an important biological role together with a range of chemical compounds produced in laticifers, probably acting synergistically [4,32]. ...
... rutin, quercetin, luteolin), phenolic acids (gallic acid, chlorogenic acid), unsaturated fatty acids (linoleic acid, oleic acid), carotenoids, saponins, and some others [71,77,78]. Recent data also showed the rich protein content of the milky sap, connected with defense response and generation of precursor metabolites and energy [22][23][24]74]. Predominant proteins contained in C. majus latex include major latex protein (MLP, presumably belonging to Bet v1-like superfamily), polyphenol oxidase (PPO), peroxidase (POX), lipoxygenase (LOX), and enzymes responsible for phenylpropanoid and alkaloid biosynthesis [24,25]. ...
... Additionally, the occurrence of highly conserved benzylisoquinoline alkaloid biosynthetic enzymes in plants with and without latex (e.g. Chelidonium majus with latex comparing to Corydalis cava without it) suggests that laticifers arose in the Papaveraceae after the emergence of the metabolic pathways [23,56]. Laticiferous Papaveraceae accumulate and sequestrate preexisting alkaloids in laticifers. ...
The aim of this review is to cover most recent research on plant pathogenesis- and defense-related proteins from latex-bearing medicinal plant Chelidonium majus (Papaveraceae) in the context of its importance for latex activity, function, pharmacological activities, and antiviral medicinal use. These results are compared with other latex-bearing plant species and recent research on proteins and chemical compounds contained in their latex. This is the first review, which clearly summarizes pathogenesis-related (PR) protein families in latex-bearing plants pointing into their possible functions. The possible antiviral function of the latex by naming the abundant proteins present therein is also emphasized. Finally latex-borne defense system is hypothesized to constitute a novel type of preformed immediate defense response against viral, but also non-viral pathogens, and herbivores.
... Plants may use the Hsp90/Hsp70 chaperone machinery to facilitate the process involved in cell-to-cell transport of macromolecules. The presence of Hsp90 protein was previously reported for C. majus shoots (Aoki et al., 2002;Nawrot et al., 2014). Together with other up-regulated heat shock proteins, it functions in the stabilization of proteins and membranes and can assist in protein refolding under stress conditions (Qureshi et al., 2007;Sangster and Queitsch, 2005). ...
... Plants are vulnerable to increase of environmental stresses, which result in excessive accumulation of reactive oxygen species (ROS), causing cell damage. The presence of redox proteins was previously reported for C. majus shoots and latex (Nawrot et al., 2016(Nawrot et al., , 2007b(Nawrot et al., , 2014. ROS scavenging enzymes were also up-regulated in the early growth stage and down-regulated in the mature stage of Panax ginseng C.A. Meyer (Ma et al., 2016). ...
... They have been shown to confer resistance to viruses, fungi, bacteria, aphids, and even nematodes (Fourmann et al., 2001). Such proteins were also identified in Corydalis cava tubers (Nawrot et al., 2014). NBS-LRR and CC-NBS-LRR homologs were also up-regulated over the growth period in P. ginseng, what could improve its disease resistance (Ma et al., 2016). ...
Chelidonium majus L. (Papaveraceae) latex is used in traditinonal folk medicine to treat papillae, warts, condylomas, which are visible effects of human papilloma virus (HPV) infections. The aim of this work was to provide new insights into the biology and medicinal use of C. majus milky sap in the flowering and fruit ripening period of the plant by comparing the protein content between samples collected on respective developmental stages using LC-MS-based label-free proteome approach. For quantification, the multiplexed LC-MS data were processed using comparative chemometric approach. Progenesis LC-MS results showed that in green fruit phase (stage IV), comparing to flowering phase (stage III) of plant development, a range of proteins with higher abundance were identified as stress- and defense-related. On the other hand at stage III very intense protein synthesis, processes of transcription, protein folding and active transport of molecules (ABC transporters) are well represented. 2-DE protein maps showed an abundant set of spots with similar MWs (about 30–35 kDa) and pIs (ca. 5.5–6.5), which were identified as major latex proteins (MLPs). Therefore we suggest that biological activity of C. majus latex could be related to its protein content, which shifts during plant development from intense biosynthetic processes (biosynthesis and transport of small molecules, like alkaloids) to plant defense mechanisms against pathogens. Further studies will help to elucidate if these defense-related and pathogenesis-related proteins, like MLP, together with small-molecule compounds, could inhibit viral infection, what could be a step to fully understand the medicinal activity of C. majus latex.
... Proteins were identified by MS/MS via information-dependent acquisition of fragmentation spectra (Nawrot et al. 2007a(Nawrot et al. , 2014 using MASCOT 2.4.1 search (Perkins et al. 1999; Matrix Science, London, UK; www.matrixscience. com) against C. majus CDS database. ...
... For proteomic assessments using the novel database, we used two types of datasets. One of them was previously published data for proteomic content of C. majus whole plant extracts using an approach of protein separation by 1-D SDS-PAGE to subsequent LC-MS/MS analysis of individual gel bands (i.e., shotgun approach) (Nawrot et al. 2014). The main advantages of such approach are sample purity after SDS-PAGE improving proteome coverage of analyzed samples (Schulze and Usadel 2010;Matros et al. 2011). ...
Main conclusion:
A novel annotated Chelidonium majus L. transcriptome database composed of 23,004 unique coding sequences allowed to significantly improve the sensitivity of proteomic C. majus assessments, which showed novel defense-related proteins characteristic to its latex. To date, the composition of Chelidonium majus L. milky sap and biosynthesis of its components are poorly characterized. We, therefore, performed de novo sequencing and assembly of C. majus transcriptome using Illumina technology. Approximately, 119 Mb of raw sequence data was obtained. Assembly resulted in 107,088 contigs, with N50 of 1913 bp and N90 of 450 bp. Among 34,965 unique coding sequences (CDS), 23,004 obtained CDS database served as a basis for further proteomic analyses. The database was then used for the identification of proteins from C. majus milky sap, and whole plant extracts analyzed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) approach. Of about 334 different putative proteins were identified in C. majus milky sap and 1155 in C. majus whole plant extract. The quantitative comparative analysis confirmed that C. majus latex contains proteins connected with response to stress conditions and generation of precursor metabolites and energy. Notable proteins characteristic to latex include major latex protein (MLP, presumably belonging to Bet v1-like superfamily), polyphenol oxidase (PPO, which could be responsible for browning of the sap after exposure to air), and enzymes responsible for anthocyanidin, phenylpropanoid, and alkaloid biosynthesis.
... Dentro de los sistemas de defensa de organismos tales como insectos, anfibios, mamíferos y plantas se pueden destacar los péptidos antimicrobianos (AMP por el inglés Antimicrobial Peptides). Han sido identificados en una amplia variedad de organismos, incluyendo arqueas y bacterias, lo que deja entrever su presencia temprana en la evolución (Nawrot et al., 2014;Rustagi et al., 2014;Dobson et al., 2013;Ng-Choi et al., 2013;Zhu, 2007). Los AMP hacen parte de la inmunidad no especifica e innata de los hospederos, los cuales pueden ser considerados antibióticos endógenos y moléculas efectoras. ...
... Aquellos AMP estabilizados por cisteína tienen un núcleo gamma compuesto de dos láminas beta antiparalelas. Otros péptidos tienen estructuras basadas en alfa hélices, o carecen de una estructura secundaria uniforme (Nawrot et al., 2014;Dobson et al., 2013;Ng-Choi et al., 2014;Park et al., 2007;Brogden, 2005;Ganz & Lehrer, 1999). ...
Los sistemas de defensa de los organismos vivientes abarcan una gran gama de biomoléculas, un importante grupo de estas, son los péptidos antimicrobianos (AMP del inglés Antimicrobial Peptides). Estas péptidos forman parte de la inmunidad no específica e innata de los seres vivos y son activos contra patógenos(bacterias, hongos, virus y protozoarios). Las plantas, al ser una fuente de alimento, son atacadas pormuchas clases de microorganismos, aunque solo unos pocos les pueden causar enfermedades. Las plantas y los organismos patógenos han coevolucionado, así las primeras han logrado estrategias efectivas de resistirel ataque de sus invasores. Las proteínas relacionadas con patogénesis (PR) hacen parte de un mecanismo inducible de resistencia, las cuales se expresanen gran proporción, en tejidos vegetales parasitados. Las PR tienen un peso molecular de entre 5 a 75 kDa, aquellas con pesos inferiores a 10 kDa se denominanAMP, entre las que se cuentan las quitinasas, endoproteasas, peroxidasas, heveinas, oxalato oxidasas, proteínas de transferencia de lípidos (LTP del inglés Lipid Transfer Protein o Lipid Transfer Peptide) y defensinas,entre otras. Las LTP presentan un tamaño de 7-10 kDa, se caracterizan por participar en intercambio de lípidos entre membranas, ser ricas en residuosde cisteína y por poseer una conformación cationica. Las LTP tienen capacidad de inhibir el crecimiento de bacterias y de hongos por su interacción con fosfolípidos de la membrana del patógeno y también por lacompetencia con moléculas señalizadoras del agente invasor por el receptor de la planta, y así la LTP evitaría el reconocimiento planta-patógeno. Aquí se presentan algunas consideraciones importantes acercade esta temática y su perspectiva en la agricultura comercial colombiana.
... Dentro de los sistemas de defensa de organismos tales como insectos, anfibios, mamíferos y plantas se pueden destacar los péptidos antimicrobianos (AMP por el inglés Antimicrobial Peptides). Han sido identificados en una amplia variedad de organismos, incluyendo arqueas y bacterias, lo que deja entrever su presencia temprana en la evolución (Nawrot et al., 2014;Rustagi et al., 2014;Dobson et al., 2013;Ng-Choi et al., 2013;Zhu, 2007). Los AMP hacen parte de la inmunidad no especifica e innata de los hospederos, los cuales pueden ser considerados antibióticos endógenos y moléculas efectoras. ...
... Aquellos AMP estabilizados por cisteína tienen un núcleo gamma compuesto de dos láminas beta antiparalelas. Otros péptidos tienen estructuras basadas en alfa hélices, o carecen de una estructura secundaria uniforme (Nawrot et al., 2014;Dobson et al., 2013;Ng-Choi et al., 2014;Park et al., 2007;Brogden, 2005;Ganz & Lehrer, 1999). ...
Defense mechanisms in biological organisms are composed by biomolecules, an important group is the antimicrobial peptides (AMPs). AMPs are part of the non-specific and innate immunity with activity against pathogens (bacteria, fungi, viruses and protozoa). Plants (a food source) are attacked by microorganisms, although a few cause disease. Plants and pathogens have coevolved, so plants have effective resistance strategies. Pathogenesis-related proteins (PR) are part of an inducible mechanism of resistance; PRs are in high proportion in parasitized plant tissues. PRs have a molecular weight of 5-75 kDa, AMPs have less than 10 kDa including chitinases, endoproteases, peroxidases, heveinas, oxalate oxidases, lipid transfer protein (LTP) and defensins among others. LTPs have a molecular weight of 7-10 kDa and contribute with lipid exchange between membranes; LTPs contain conserved cysteine residues and have a cationic conformation. LTPs inhibit bacteria and fungi growth by interaction with membrane phospholipids of pathogens and competition for plant receptor avoiding plant-pathogen recognition. Here, we show important considerations about this topic and perspectives in colombian commercial crops.
... AMPs interact with the phospholipids plasma membrane and other intracellular or extracellular sites to prevent the microbial attack [106]. A few well-characterized AMPs are snakins, thionins and defensins [107]. A few studies have shown that AMPs form pores in the membrane, resulting in the leakage of ions and metabolites or depolarization. ...
Plants are essential for humans as they serve as a source of food, fuel, medicine, oils, and more. The major elements that are utilized for our needs exist in storage organs, such as seeds. These seeds are rich in proteins, show a broad spectrum of physiological roles, and are classified based on their sequence, structure, and conserved motifs. With the improvements to our knowledge of the basic sequence and our structural understanding, we have acquired better insights into seed proteins and their role. However, we still lack a systematic analysis towards understanding the functional diversity associated within each family and their associations with allergy. This review puts together the information about seed proteins, their classification, and diverse functional roles along with their associations with allergy.
... Chelidonium majus belongs to the Papaveraceae family, and it is widely found worldwide, including Asia, Europe, North America, and Northern Africa [1]. The aerial parts of this plant consist of isoquinoline alkaloids (berberine, chelerythrine, chelidonine, coptisine, sangui-narine, and stylopine), whereas herbs of C. majus consist of carotenoids, flavonoids, organic acids, and proteins [2][3][4][5][6][7]. The extract of C. majus has various antioxidant properties (elimination of reactive oxygen species (ROS), and protection from oxidative stress), with a wide range of medicinal properties such as analgesic, anti-inflammatory, antimicrobial, anti-spasmodic, antitumor, antiviral, choleretic, and hepatoprotective properties [1,2,5,[8][9][10][11][12]. It has also been used for the treatment of asthma, cancer, chronic bronchitis, eczema, gas- ...
Chelidonium majus L. is a perennial herbaceous plant that has various medicinal properties. However, the genomic information about its carotenoid biosynthesis pathway (CBP), xanthophyll biosynthesis pathway (XBP), and apocarotenoid biosynthesis pathway (ABP) genes were limited. Thus, the CBP, XBP, and ABP genes of C. majus were identified and analyzed. Among the 15 carotenoid pathway genes identified, 11 full and 4 partial open reading frames were determined. Phylogenetic analysis of these gene sequences showed higher similarity with higher plants. Through 3D structural analysis and multiple alignments, several distinct conserved motifs were identified, including dinucleotide binding motif, carotene binding motif, and aspartate or glutamate residues. Quantitative RT-PCR showed that CBP, XBP, and ABP genes were expressed in a tissue-specific manner; the highest expression levels were achieved in flowers, followed by those in leaves, roots, and stems. The HPLC analysis of the different organs showed the presence of eight different carotenoids. The highest total carotenoid content was found in leaves, followed by that in flowers, stems, and roots. This study provides information on the molecular mechanisms involved in CBP, XBP, and ABP genes, which might help optimize the carotenoid production in C. majus. The results could also be a basis of further studies on the molecular genetics and functional analysis of CBP, XBP, and ABP genes.
... The resulting powder was dissolved in 0.1 M Tris-Cl buffer, pH 8.0, containing 10% glicerol (extract:buffer ratio was 1:1). The tuber extract (50% v/v) samples were separated into a supernatant, and a pellet fraction, by centrifugation at 12000 rpm for 20 min at 4 • C as described in the protocol [56], with modifications, and proteins from the supernatant were purified according to Diffley and Stillman [57]. Samples were stored at − 20 • C for further analysis. ...
Thaumatin-like proteins (TLPs, osmotins) form a protein family which shares a significant sequence homology to the sweet-tasting thaumatin from the plant Thaumatococcus daniellii. TLPs are not sweet-tasting and are involved in response to biotic stresses and developmental processes. Recently it has been shown using a proteomic approach that the tuber extract from Corydalis cava (Papaveraceae) contains a TLP protein. The aim of this work was to characterize the structure and expression of TLP from C. cava tubers. The results obtained using a PCR approach with degenerate primers demonstrated a coding sequence of a novel protein, named CcTLP1. It consists of 225 aa, has a predicted molecular weight of 24.2 kDa (NCBI GenBank accession no. KJ513303) and has 16 strictly conserved cysteine residues, which form 8 disulfide bridges and stabilize the 3D structure. CcTLP1 may be classified into class IX of plant TLPs. The highest CcTLP1 expression levels were shown by qPCR in the stem of the plant compared to other organs and in the medium-size plants compared to other growth phases. The results confirm that CcTLP1 is expressed during plant growth and development until flowering, with a possible defensive function against different stress conditions.
... The aerial part of this plant contains isoquinoline alkaloids, such as chelidonine, chelerythrine, sanguinarine, berberine, coptisine and stylopine [4,7,8]. Moreover, this herb includes organic acids, carotenoids, flavonoids and proteins [9][10][11]. Studies showed that methanolic extract from the herb of CM significantly suppressed the progression of collagen-induced arthritis (mice model) and that this action was characterized by the decreased production of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), interferon γ (IFN-γ), B cells, γδ T cells (in spleen) and increased proportion of CD4+CD25+ regulatory T cells in vivo. ...
The aim of the study was to evaluate analgesic activity (“hot plate” test), anti-inflammatory activity
(carrageenan-induced paw edema) and locomotor activity in rats under the influence of three fractions
of Chelidonium majus herb extract: full water extract (FWE), protein enriched fraction (PEF), and
non-protein fraction (NPF). Effects of the fractions on the level of chosen cytokines and their mRNA
levels were also assessed using lipopolysaccharide (LPS) administration as a proinflammatory cue.
All fractions and diclofenac did not affect the locomotor activity of rats in comparison with the control
group. FWE and PEF three hours after administration showed statistically significant analgesic activities
comparable to morphine (p < 0.05). A slight reduction in rat paw edema was observed after three
(comparable with diclofenac) and six hours in the NPF group. FWE revealed a statistically significant
pro-inflammatory effect after three hours in comparison with the control group. Peripheral IL-1 and
IL-4 cytokine concentrations were reduced under FWE and NPF, PEF fractions. The combination
of FWE, PEF and NPF together with LPS showed only the effects of LPS. We suggest that protein
enriched fraction (PEF) produced centrally mediated (morphine-like) analgesic action, whereas the
anti-inflammatory potential was shown only after LPS-induced inflammation. The precise mechanisms
involved in the production of anti-nociceptive and anti-inflammatory responses of studied fractions
are not completely understood, but they may be caused rather by the presence of protein more than
alkaloids-enriched fraction. This fraction of the extract could be used as an alternative therapy for the
prevention of inflammatory-related diseases in the future, but further studies are needed
... Chelidonium majus Chelidonium majus L. (Family: Papaveraceae), is an important plant which has been used for the treatment of many diseases in different part of Western Europe, and in Chinese herbal medicines for centuries. It has multiple applications in folk medicine because of its antitumoral, cytotoxic, anti-inflammatory and antimicrobial activities (Saglam and Arar, 2003;Lanvers-Kaminsky et al., 2006;Biswas et al., 2008;Kulp and Bragina, 2013) and has recently been reported to contain different pathogenesisrelated and low molecular inducible antimicrobial peptides (Nawrot et al., 2014). Reports showed that the crude extracts and its main components-isoquinoline, other alkaloids (such as sanguinarine, chelidonine, chelerythrine, berberine, protopine and coptisine), flavonoids and phenolic acids contain anti-inflammatory, antioxidant, antimicrobial, immunomodulatory, antitumoral and many other therapeutic properties (Jiang and Dusting, 2003;Palombo, 2006;Talhouk et al., 2007;Nadova et al., 2008;Zuo et al., 2008;Cahlíkova et al., 2010;Gilca et al., 2010, Kulp et al., 2011Yao et al., 2011;Zhang et al., 2011;Koriem et al., 2013;Kuenzel et al., 2013). ...
Radiation exposure leads to several pathophysiological conditions, including oxidative damage, inflammation and fibrosis, thereby affecting the survival of organisms. This review explores the radiation countermeasure properties of fourteen (14) plant extracts or plant-derived compounds against these cellular manifestations. It was aimed at evaluating the possible role of plants or its constituents in radiation countermeasure strategy. All the 14 plant extracts or compounds derived from it and considered in this review have shown some radioprotection in different in vivo, ex-vivo and or in vitro models of radiological injury. However, few have demonstrated advantages over the others. C. majus possessing antioxidant, anti-inflammatory and immunomodulatory effects appears to be promising in radioprotection. Its crude extracts as well as various alkaloids and flavonoids derived from it, have shown to enhance survival rate in irradiated mice. Similarly, curcumin with its antioxidant and the ability to ameliorate late effect of radiation exposure, combined with improvement in survival in experimental animal following irradiation, makes it another probable candidate against radiological injury. Furthermore, the extracts of P. hexandrum and P. kurroa in combine treatment regime, M. piperita, E. officinalis, A. sinensis, nutmeg, genistein and ginsan warrants further studies on their radioprotective potentials. However, one that has received a lot of attention is the dietary flaxseed. The scavenging ability against radiation-induced free radicals, prevention of radiation-induced lipid peroxidation, reduction in radiation cachexia, level of inflammatory cytokines and fibrosis, are some of the remarkable characteristics of flaxseed in animal models of radiation injury. While countering the harmful effects of radiation exposure, it has shown its ability to enhance survival rate in experimental animals. Further, flaxseed has been tested and found to be equally effective when administered before or after irradiation, and against low doses (≤5 Gy) to the whole body or high doses (12-13.5 Gy) to the whole thorax. This is particularly relevant since apart from the possibility of using it in pre-conditioning regime in radiotherapy, it could also be used during nuclear plant leakage/accidents and radiological terrorism, which are not pre-determined scenarios. However, considering the infancy of the field of plant-based radioprotectors, all the above-mentioned plant extracts/plant-derived compounds deserves further stringent study in different models of radiation injury.
The collection of the Tenth International Scientific and Practical Conference “Medicinal Herbs: from past experience to new technologies” in a separate section the memories, photo materials and bibliography of Lidia Panasivna Shelud’ko, a famous breeder and researcher of medicinal plants, are collected on the occasion of her 85th birthday, and presents the results of the investigations of medicinal plants, especially their introduction, biology, breeding, physiology and phytochemistry,
propagation and cultivation, pharmacy, use in agriculture and industry.
Since time immemorial, humans utilize plants for various needs such as food, shelter, fuelwood, and medicine. Plants exhibit medicinal properties due to the presence of several classes of phytochemicals, especially secondary metabolites, also known as specialized metabolites (SMs). Plants are chemical factories and many important drugs used today are derived from the plants or plant-inspired semi-biosynthetic pathways. Large numbers of plants are being increasingly explored for medicinally important specialized metabolites because there has been an increase in the interest in bioprospecting natural sources of more drugs. However, current bioprospecting approaches are not sustainable. Therefore, modern approaches can be used for the characterization of medicinally important plants for metabolites/phytochemicals present in them and for the validation of their medicinal properties in treating several serious diseases. Recent decades have seen the emergence of new technologies which can be deployed to characterize the phytochemicals present in the plants on a large scale. The genetic biosynthetic pathways of the plants can also be traced using high-throughput technologies such as genomics, proteomics, metabolomics, and transcriptomics. Several studies aim to characterize biosynthetic pathways of phytochemicals, and efforts are on identifying genes, proteins, and metabolomes associated with a particular metabolite. All this has led to an emergence of an entirely new discipline known as phytochemical genomics, which involves the integration of multi-omics approaches, such as genomics, transcriptomics, proteomics, and metabolomics intending to decipher gene-protein-metabolite networks. This chapter introduces the concept of phytochemical genomics, various techniques/technologies, and approaches that are used in phytochemical genomics and some of the examples of medicinal plants, where these technologies have been successfully utilized. This chapter further delves into linking gene editing technologies with omics information for the improvement of medicinal plants.
Chelidonium genus (Papaveraceae) is widely distributed in the world. The only species of the genus is Chelidonium majus. The drug and the herb mainly carry isoquinoline alkaloids of the benzophenanthridine type; some of the alkaloids it carries are chelidonine, chelerythrine, and sanguinarine; and protopine can be listed as protoberberine, berberine, stylopine, and protopine. All parts of the plant carry isoquinoline-type alkaloids in varying amounts.Chelidonium majus is a drug registered in the European Pharmacopoeia under the monograph name Chelidonii herba. The European Pharmacopoeia standardizes the drug on the total alkaloids calculated on the chelidonine and requires a minimum of 0.6% alkaloids in aerial parts. The amount of alkaloids is quite high in the latex of the plant. This latex, rich in alkaloids, turns orange-red when it comes into contact with air.The first medical records on the plant date back to the time of Dioscorides. Latex has a strong antiviral effect and is used in the treatment of warts. The tinctures prepared from aerial parts are used as antispasmodic and cholagogues in Europe. Until now, extracts of C. majus and a few isolated compounds have been determined to be primarily antispasmodic and cholekinetic. Apart from these, it has been determined that the extract and the isolated alkaloids have anticancer properties and are effective against pathogenic microorganisms. Chelidonium majus and isolated alkaloids show anti-inflammatory effects. In this study, literature data on Chelidonium majus were investigated, and phytochemical and phytotherapeutic properties of the plant were discussed.KeywordsGreater celandine
Chelidonium majus
EthnomedicinalBioactive compositionIn vitro and in vivo studiesToxicityMode of actionCommercial formulations
BACKGROUND
plant viral diseases are difficult to control and have caused serious damage to agricultural industry. Nowadays, botanical biopesticide characterized by environment friendly, safe to non‐target organism and not as susceptible to produce drug resistance, have exhibited great potential to be developed as antiviral agents. To screen the natural products with antiviral effect, three alkaloids possessed anti‐TMV activity were isolated from Chelidonium majus. And the modes of action were investigated.
RESULT
The anti‐TMV effect of crude extracts at 10 mg ml‐1 was 51.73%. Bioassay‐guided fractionation and isolation of the compounds with anti‐TMV activity were performed on the methanol extract of C. majus yielding three bioactive alkaloids namely: chelerythrine (1), chelidonine (2), and sanguinarine (3). The results of bioassay showed that chelerythrine exhibited great inactivation, proliferation inhibition and protection effects against TMV at 0.5 mg ml‐1 with the efficiency of 72.67%, 77.52% and 59.34%, respectively. Chelidonine at 0.1 mg ml‐1 can provide 54.90% and 64.45% inhibitions on TMV through inducing resistance in two kinds of tobacco. Sanguinarine showed a weaker protection for resisting TMV in comparison to chelerythrine and chelidonine.
CONCLUSION
Chelerythrine and chelidonine displayed significant inhibitions on TMV with different modes of action. These results provided important evidence that the extracts in C. majus might be a potential source of new drugs in controlling virus disease agriculturally.
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In this review we have collected information regarding latex proteins and their contribution to latex-borne defense among a variety of latex-bearing plants. We comprehensively describe the functions and properties of different pathogenesis- and defense-related proteins found in plant latex, including peroxidases, lipoxygenases, polyphenol oxidases, major latex proteins, β-1,3-glucanases, chitinases, osmotins, proteases, and others. The last section describes proteomic changes occurring during plant-virus interactions in latex-bearing papaya infected with Papaya meleira virus as an example.
Plant latex is a milky-like fluid (sap) that is stored in plant specialized cells – laticifers. It contains a mixture of phytochemicals, proteins and enzymes, such as alkaloids, phenolics, terpenoids, defense proteins, proteases and chitinases. Most of these latex ingredients possess pharmacologic activity. Due to its curing effects, Chelidonium majus latex has been used in traditional folk medicine to treat skin warts and tubercles. Nowadays, natural plant latex derived molecules and their synthetic analogs are used in medicine as antimicrobial agents, analgesics, antiproliferative and anticancer drugs. Moreover, it has been reported that alkaloids and phenolics from the plant latex exhibit potential therapeutic effects against neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Most of natural plant latex derived products are present in low amounts, difficult to isolate, possess poor bioavailability and stability. Recent advancements in nanotechnology offer advanced nanomaterials as nanocarriers of pharmacologically active latex natural agents. Conjugation of small natural latex compounds with nanoparticles, liposomes, micelles, nanodiamonds and carbon nanotubes allows to improve bioavailability and stability of these natural pharmacologically active latex compounds. Furthermore, nanoscience approach provides time-controlled and site-specific delivery of potential therapeutics from latex, what gives new promising perspectives for their future use in medicine.
As antique as Dioscorides era are the first records on using Chelidonium as a remedy to several sicknesses. Inspired by the “signatura rerum” principle and an apparent ancient folk tradition, various indications were given, such as anti-jaundice and cholagogue, pain-relieving, and quite often mentioned—ophthalmological problems. Central and Eastern European folk medicine has always been using this herb extensively. In this region, the plant is known under many unique vernacular names, especially in Slavonic languages, associated or not with old Greek relation to “chelidon”—the swallow. Typically for Papaveroidae subfamily, yellow-colored latex is produced in abundance and leaks intensely upon injury. Major pharmacologically relevant components, most of which were first isolated over a century ago, are isoquinoline alkaloids—berberine, chelerythrine, chelidonine, coptisine, sanguinarine. Modern pharmacology took interest in this herb but it has not ended up in gaining an officially approved and evidence-based herbal medicine status. On the contrary, the number of relevant studies and publications tended to drop. Recently, some controversial reports and sometimes insufficiently proven studies appeared, suggesting anticancer properties. Anticancer potential was in line with anecdotical knowledge spread in East European countries, however, in the absence of directly-acting cytostatic compounds, some other mechanisms might be involved. Other properties that could boost the interest in this herb are antimicrobial and antiviral activities. Being a common synanthropic weed or ruderal plant, C. majus spreads in all temperate Eurasia and acclimates well to North America. Little is known about the natural variation of bioactive metabolites, including several aforementioned isoquinoline alkaloids. In this review, we put together older and recent literature data on phytochemistry, pharmacology, and clinical studies on C. majus aiming at a critical evaluation of state-of-the-art from the viewpoint of historical and folk indications. The controversies around this herb, the safety and drug quality issues and a prospective role in phytotherapy are discussed as well.
The tribe Chelidonieae of family Papaveraceae is a botanical source of various medicinal components, which has been commonly used in traditional Chinese medicine for many centuries. Increasing interest in Chelidonieae pharmaceutical resources has led to additional discoveries of alkaloids, flavonoids, terpenoids, glycosides, and many other compounds in various Chelidonieae species and to investigations on their chemotaxonomy, molecular phylogeny, and pharmacology. In continuation with our studies on Chelidonieae pharmacophylogeny, we review the phytochemistry, pharmacology, chemotaxonomy, and molecular phylogeny of Chelidonieae and their relevance to drug efficacy. Literature search is used to characterize the global scientific endeavor in the versatile technologies being used. The most remarkable pharmacological effect of both Chelidonieae compounds and extracts is the anticancer activity. The congruence between chemotaxonomy and molecular phylogeny is revealed and the evolution of plant alkaloid biosynthesis discussed. There is a lack of genomic, transcriptomic, and metabolomic studies of Chelidonieae. For both the sustainable utilization of Chelidonieae pharmaceutical resources and finding novel compounds with potential clinical utility, systems biology and omics technologies will play an increasingly important role in future medicinal research involving bioactive compounds of Chelidonieae.
Medicinal Plants: Chemistry, Biology and Omics reviews the phytochemistry, chemotaxonomy, molecular biology, and phylogeny of selected medicinal plant tribes and genera, and their relevance to drug efficacy. Medicinal plants provide a myriad of pharmaceutically active components, which have been commonly used in traditional Chinese medicine and worldwide for thousands of years. Increasing interest in plant-based medicinal resources has led to additional discoveries of many novel compounds, in various angiosperm and gymnosperm species, and investigations on their chemotaxonomy, molecular phylogeny and pharmacology. Chapters in this book explore the interrelationship within traditional Chinese medicinal plant groups and between Chinese species and species outside of China. Chapters also discuss the incongruence between chemotaxonomy and molecular phylogeny, concluding with chapters on systems biology and "-omics? technologies (genomics, transcriptomics, proteomics, and metabolomics), and how they will play an increasingly important role in future pharmaceutical research.
The oxidative burst, the generation of reactive oxygen species (ROS) in response to microbial pathogen attack, is a ubiquitous early part of the resistance mechanisms of plant cells. It has also become apparent from the study of a number of plant–pathogen interactions and those modelled by elicitor treatment of cultured cells that there may be more than one mechanism operating. However, one mechanism may be dominant in any given species. NADPH oxidases have been implicated in a number of systems and have been cloned and characterized. However, the enzyme system which is the major source of ROS in French bean ( Phaseolus vulgaris ) cells treated with a cell wall elicitor from Colletotrichum lindemuthianum , appears to be dependent on an exocellular peroxidase. The second component, the extracellular alkalinization, occurs as a result of the Ca ²⁺ and proton influxes and the K ⁺ efflux common to most elicitation systems as one of the earliest responses. The third component, the actual reductant/substrate, has remained elusive. The low molecular weight compound composition of apoplastic fluid was compared before and after elicitation. The substrate only becomes available some min after elicitation and can be extracted, so that by comparing the profiles by LC‐MS it has been possible to identify possible substrates. The mechanism has proved to be complex and may involve a number of low molecular weight components. Stimulation of H 2 O 2 production was observed with saturated fatty acids such as palmitate and stearate without concomitant oxylipin production. This biochemical evidence is supported by immunolocalization studies on papillae forming at bacterial infection sites that show the peroxidase isoform present at sites of H 2 O 2 production revealed by cerium chloride staining together with the cross‐linked wall proteins and callose and callose synthase. The peroxidase has been cloned and expressed in Pichia pastoris and has been shown to catalyse the oxidation reaction with the same kinetics as the purified enzyme. Furthermore, Arabidopsis plants transformed heterologously using the French bean peroxidase in antisense orientation have proved to be highly susceptible to bacterial and fungal pathogens. Thus it is possible that Arabidopsis is another species with the potential to mount an apoplastic oxidative burst and these transformed plant lines may be useful to identify the peroxidase that is responsible.
Abstract In line 12 of page 1, replace "GmGER 9" with "GmGER 15".
Milky sap isolated from Chelidonium majus L. (Greater Celandine) serves as a rich source of various biologically active substances such as alkaloids, flavonoids and phenolic acids. Previous research showed that the activity of Ch. majus milky sap may depend also on the presence of biologically active proteins. The goal of this study was to evaluate the biological effect of two nucleases isolated from Ch. majus milk sap, CMN1 of 20 kDa and CMN2 of 36 kDa, on HeLa and CHO tumour cell lines. Both studied nucleases together with other proteins in the sap of the plant are involved in stress and defence reactions against different pathogens. After 48 h incubation of CMN1 and CMN2 only with HeLa cells, the dependence between the number of apoptotic lesions and the concentration of applied nuclease was observed. The highest proapoptotic activity was induced by 13.3 ng/ml concentration of CMN2 collected in May (62 +/- 3% HeLa cells were apoptotic). Moreover, the proportion of necrotic cells in all concentrations of the nucleases and both cell lines was relatively low (1-8 +/- 0.5%). In summary, results of this study show that purified nucleases CMN1 and CMN2 isolated from Ch. majus milky sap exhibit apoptotic activity in HeLa tumour cell line, but not in CHO cells, without inflammatory reaction.
Several isoforms of superoxide dismutase (SOD) with a high isoelectric point (pI) have been identified by isoelectric focusing chromatography in protein extracts from Scots pine (Pinus sylvestris) needles. One of these isoforms, a CuZn-SOD with a pI of about 10 and thus denoted hipI-SOD, has been isolated and purified to apparent homogeneity. A cDNA encoding the hipI-SOD protein was cloned and sequenced. Northern hybridization of mRNA isolated from different organs and tissues showed that hipI-SOD has a markedly different pattern of expression compared with chloroplastic and cytosolic SOD. Furthermore, the transcript levels of hipI-SOD and cytosolic SOD were found to respond differently to mechanical wounding, treatment with oxidized glutathione, paraquat, and ozone. Immunogold electron microscopy localized the hipI-SOD in the plasma membrane of sieve cells and the Golgi apparatus of albuminous cells. Moreover, high protein density was also detected in extracellular spaces such as secondary cell wall thickenings of the xylem and sclerenchyma and in intercellular spaces of parenchyma cells.
Plant antimicrobial peptides (AMPs) are a component of barrier defense system of plants. They have been isolated from roots, seeds, flowers, stems, and leaves of a wide variety of species and have activities towards phytopathogens, as well as against bacteria pathogenic to humans. Thus, plant AMPs are considered as promising antibiotic compounds with important biotechnological applications. Plant AMPs are grouped into several families and share general features such as positive charge, the presence of disulfide bonds (which stabilize the structure), and the mechanism of action targeting outer membrane structures.
The family of glycine-rich plant proteins (GRPs) is a large and complex group of proteins that share, as a common feature, the presence of glycine-rich domains arranged in (Gly)n-X repeats that are suggested to be involved in protein–protein interactions, RNA binding, and nucleolar targeting. These proteins are implicated in several independent physiological processes. Some are components of cell walls of many higher plants, while others are involved in molecular responses to environmental stress, and mediated by post-transcriptional regulatory mechanisms. The goals of this study are to identify the coding sequence of a novel glycine-rich RNA-binding protein from Chelidonium majus and to propose its structural model. DNA fragments obtained using degenerate PCR primers showed high sequence identities with glycine-rich RNA-binding protein coding sequences from different plant species. A 439-bp nucleotide sequence is identified coding for a novel polypeptide composed of 146 amino acids, designated as CmGRP1 (C. majus glycine-rich protein 1), with a calculated MW of 14,931 Da (NCBI GenBank accession no. HM173636). Using NCBI CDD and GeneSilico MetaServer, a single conserved domain, the RNA recognition motif (RRM), was detected in CmGRP1. The C-terminal region of CmGRP1 is a glycine-rich motif (GGGGxxGxGGGxxG), and it is predicted to be disordered. Based on a 1fxl crystal structure, a 3D model of CmGRP1 is proposed. CmGRP1 can be classified as a class IVa plant GRP, implicated to play a role in plant defense.
The aim of this review is to present the current state of our understanding on the structure, regulation, and function of plant pathogenesis-related protein family 10 (PR-10). This protein family consists of relatively diverse members subgrouped into classes that suggest different functions. It is believed that PR-10 proteins are involved in plant defense because their genes are usually induced upon the attack of various pathogens and by environmental stresses. However, updated evidence shows that PR-10 proteins display several additional functions, including a role in developmental processes and enzymatic activities in secondary metabolism. Because of the complexity of the PR-10 gene family and its potential multiple functions, it is important to summarize current knowledge as basis for a further dissection of the functions of PR-10 proteins and a better understanding of their structural adaptation. This paper provides the first review of existing knowledge of plant PR-10 proteins and examines their structural and functional adaptations.
Inducible defense-related proteins have been described in many plant species upon infection with oomycetes, fungi, bacteria, or viruses, or insect attack. Several types of proteins are common and have been classified into 17 families of pathogenesis-related proteins (PRs). Others have so far been found to occur more specifically in some plant species. Most PRs and related proteins are induced through the action of the signaling compounds salicylic acid, jasmonic acid, or ethylene, and possess antimicrobial activities in vitro through hydrolytic activities on cell walls, contact toxicity, and perhaps an involvement in defense signaling. However, when expressed in transgenic plants, they reduce only a limited number of diseases, depending on the nature of the protein, plant species, and pathogen involved. As exemplified by the PR-1 proteins in Arabidopsis and rice, many homologous proteins belonging to the same family are regulated developmentally and may serve different functions in specific organs or tissues. Several defense-related proteins are induced during senescence, wounding or cold stress, and some possess antifreeze activity. Many defense-related proteins are present constitutively in floral tissues and a substantial number of PR-like proteins in pollen, fruits, and vegetables can provoke allergy in humans. The evolutionary conservation of similar defense-related proteins in monocots and dicots, but also their divergent occurrence in other conditions, suggest that these proteins serve essential functions in plant life, whether in defense or not.
Molecular chaperones and foldases are a diverse group of proteins that in vivo bind to misfolded or unfolded proteins (non-native or unstable proteins) and play important role in their proper folding. Stress conditions compel altered and heightened chaperone and foldase expression activity in the endoplasmic reticulum (ER), which highlights the role of these proteins, due to which several of the proteins under these classes were identified as heat shock proteins. Different chaperones and foldases are active in different cellular compartment performing specific tasks. The review will discuss the role of the ER chaperones and foldases under stress conditions to maintain proper protein folding dynamics in the plant cells and recent advances in the field. The ER chaperones and foldases, which are described in article, are binding protein (BiP), glucose regulated protein (GRP94), protein-disulfide isomerase (PDI), peptidyl-prolyl isomerases (PPI), immunophilins, calnexin and calreticulin.
Plant antibacterial peptides have been isolated from a wide variety of species. They consist of several protein groups with different features, such as the overall charge of the molecule, the content of disulphide bonds, and structural stability under environmental stress. Although the three-dimensional structures of several classes of plant peptides are well determined, the mechanism of action of some of these molecules is still not well defined. However, further studies may provide new evidences for their function on bacterial cell wall. Therefore, this paper focuses on plant peptides that show activity against plant-pathogenic and human-pathogenic bacteria. Furthermore, we describe the folding of several peptides and similarities among their three-dimensional structures. Some hypotheses for their mechanisms of action and attack on the bacterial membrane surface are also proposed.
Corydalis cava Schweigg. & Koerte, the plant of numerous pharmacological activities, together with the studied earlier by our group Chelidonium majus L. (Greater Celandine), belong to the family Papaveraceae. The plant grows in Central and South Europe and produces the sizeable subterraneous tubers, empty inside, which are extremely resistant to various pathogen attacks. The Corydalis sp. tubers are a rich source of many biologically active substances, with the extensive use in European and Asian folk medicine. They have analgetic, sedating, narcotic, anti-inflammatory, anti-allergic and anti-tumour activities. On the other hand, there is no information about possible biological activities of proteins contained in Corydalis cava tubers.
Nucleolytic proteins were isolated from the tubers of C. cava by separation on a heparin column and tested for DNase activity. Protein fractions showing nucleolytic activity were tested for cytotoxic activity in human cervical carcinoma HeLa cells. Cultures of HeLa cells were conducted in the presence of three protein concentrations: 42, 83 and 167 ng/ml during 48 h. Viability of cell cultures was appraised using XTT colorimetric test. Protein fractions were separated and protein bands were excised and sent for identification by mass spectrometry (LC-ESI-MS/MS).
The studied protein fractions showed an inhibiting effect on mitochondrial activity of HeLa cells, depending on the administered dose of proteins. The most pronounced effect was obtained with the highest concentration of the protein (167 ng/ml) - 43.45 ± 3% mitochondrial activity of HeLa cells were inhibited. Mass spectrometry results for the proteins of applied fractions showed that they contained plant defense- and pathogenesis-related (PR) proteins.
The cytotoxic effect of studied proteins toward HeLa cell line cells has been evident and dependent on increasing dose of the protein. The present study, most probably, represents the first investigations on the effect of purified PR proteins from tuber extracts of a pharmacologically active plant on cell lines.
Germin and germin-like proteins constitute a ubiquitous family of plant proteins. A role of some family members in defense against pathogen attack had been proposed based on gene regulation studies and transgenic approaches. Soybean (G. max L. Merr.) germin genes had not been characterized at the molecular and functional levels.
In the present study, twenty-one germin gene members in soybean cultivar 'Maple Arrow' (partial resistance to Sclerotinia stem rot of soybean) were identified by in silico identification and RACE method (GmGER 1 to GmGER 21). A genome-wide analyses of these germin-like protein genes using a bioinformatics approach showed that the genes located on chromosomes 8, 1, 15, 20, 16, 19, 7, 3 and 10, on which more disease-resistant genes were located on. Sequence comparison revealed that the genes encoded three germin-like domains. The phylogenetic relationships and functional diversity of the germin gene family of soybean were analyzed among diverse genera. The expression of the GmGER genes treated with exogenous IAA suggested that GmGER genes might be regulated by auxin. Transgenic tobacco that expressed the GmGER 15 [corrected] gene exhibited high tolerance to the salt stress. In addition, the GmGER mRNA increased transiently at darkness and peaked at a time that corresponded approximately to the critical night length. The mRNA did not accumulate significantly under the constant light condition, and did not change greatly under the SD and LD treatments.
This study provides a complex overview of the GmGER genes in soybean. Phylogenetic analysis suggested that the germin and germin-like genes of the plant species that had been founded might be evolved by independent gene duplication events. The experiment indicated that germin genes exhibited diverse expression patterns during soybean development. The different time courses of the mRNAs accumulation of GmGER genes in soybean leaves appeared to have a regular photoperiodic reaction in darkness. Also the GmGER genes were proved to response to abiotic stress (such as auxin and salt), suggesting that these paralogous genes were likely involved in complex biological processes in soybean.
Chelidonium majus L. (family Papaveraceae), or greater celandine, is an important plant in western phytotherapy and in traditional Chinese medicine. Crude extracts of C. majus as well as purified compounds derived from it exhibit a broad spectrum of biological activities (antiinflammatory, antimicrobial, antitumoral, analgesic, hepatoprotective) that support some of the traditional uses of C. majus. However, herbal medicine also claims that this plant has several important properties which have not yet been scientifically studied: C. majus is supposed to have diuretic, antitussive and eye-regenerative effects. On the other hand, C. majus also has scientifically proven effects, e.g. anti-osteoporotic activity and radioprotection, which are not mentioned in traditional sources. Moreover, recent controversy about the hepatoprotective versus hepatotoxic effects of Chelidonium majus has renewed the interest of the medical community in this plant. This review is intended to integrate traditional ethno-medical knowledge and modern scientific findings about C. majus in order to promote understanding of its therapeutic actions as well as its toxic potential.
The lipid components of pathogen cell membranes have been considered as a poor pharmacological target,due to their universal distribution and apparent homogeneity throughout living organisms. Among the rareexceptions to this view one could mention polyene antibiotics such as amphotericin, or peptide antibioticssuch as the polymyxins and the gramicidins. In the last two decades, however, the above notion has beenchallenged by two main lines of discovery; first, natural antimicrobial peptides (AMPs) that kill pathogensby interaction with phospholipids and membrane permeabilization, and secondly, cell-penetrating peptides(CPPs), capable of introducing into cells a variety of cargoes in the absence of specific receptors, again byinteraction at some point with membrane phospholipids. For both AMPs and CPPs, the pharmacologicalproof-of-concept has been successfully demonstrated, and promising applications as nanobiotechnologicaltools have been envisaged though not hitherto materialized in clinical settings. In this review we brieflyexamine the pros and cons of these two classes of therapeutic agents, as well as strategies aimed atrationalizing and expanding their potentiality.
Mass-spectrometry-based proteomics, the large-scale analysis of proteins by mass spectrometry, has emerged as a new technology over the last decade and become routine in many plant biology laboratories. While early work consisted merely of listing proteins identified in a given organ or under different conditions of interest, there is a growing need to apply comparative and quantitative proteomics strategies toward gaining novel insights into functional aspects of plant proteins and their dynamics. However, during the transition from qualitative to quantitative protein analysis, the potential and challenges will be tightly coupled. Several strategies for differential proteomics that involve stable isotopes or label-free comparisons and their statistical assessment are possible, each having specific strengths and limitations. Furthermore, incomplete proteome coverage and restricted dynamic range still impose the strongest limitations to data throughput and precise quantitative analysis. This review gives an overview of the current state of the art in differential proteomics and possible strategies in data processing.
The strawberry Fra a 1 allergen is a homolog of the major birch pollen allergen Bet v 1. It is synthesized by red ripe fruits of Fragaria x ananassa while white fruits of a mutant genotype, which is known to be tolerated by individuals affected by allergy, are devoid of it. Proteomic analyses have shown that Fra a 1 is down-regulated in the tolerated white-fruited genotype along with enzymes of the anthocyanin pigment pathway. In this study, we report the spatial and temporal expression of three Fra a genes that encode different isoforms, and the transient RNAi-mediated silencing of the Fra a genes in strawberry fruits of the red-fruited cultivar Elsanta with an ihpRNA construct. As a consequence of reduced levels of Fra a mRNAs, fruits were obtained that produced significantly decreased levels of anthocyanins and upstream metabolites. This effect is consistent with the parallel down-regulation of the phenylalanine ammonia lyase (FaPAL) and to a lesser extent of the chalcone synthase (FaCHS) transcript levels also found in these fruits. In naturally occurring white-fruited genotypes of F. chiloensis and F. vesca, Fra a transcript levels are higher than those of the red-fruited varieties, likely to compensate for the low expression levels of FaPAL and FaCHS in these mutant genotypes. The results demonstrate that Fra a expression is directly linked to flavonoid biosynthesis and show that the Fra a allergen has an essential biological function in pigment formation in strawberry fruit.
Receptor-like kinases (RLKs) in plants are a large superfamily of proteins that are structurally similar. RLKs are involved in a diverse array of plant responses including development, growth, hormone perception and the response to pathogens. Current studies have focused attention on plant receptor-like kinases as an important class of sentinels acting in plant defense responses. RLKs have been identified that act in both broad-spectrum, elicitor-initiated defense responses and as dominant resistance (R) genes in race-specific pathogen defense. Most defense-related RLKs are of the leucine-rich repeat (LRR) subclass although new data are highlighting other classes of RLKs as important players in defense responses. As our understanding of RLK structure, activation and signaling has expanded, the role of the ubiquitin/proteasome system in the regulation of these receptors has emerged as a central theme.
Several isoforms of superoxide dismutase (SOD) with a high isoelectric point (pI) have been identified by isoelectric focusing chromatography in protein extracts from Scots pine (Pinus sylvestris) needles. One of these isoforms, a CuZn-SOD with a pI of about 10 and thus denoted hipI-SOD, has been isolated and purified to apparent homogeneity. A cDNA encoding the hipI-SOD protein was cloned and sequenced. Northern hybridization of mRNA isolated from different organs and tissues showed that hipI-SOD has a markedly different pattern of expression compared with chloroplastic and cytosolic SOD. Furthermore, the transcript levels of hipI-SOD and cytosolic SOD were found to respond differently to mechanical wounding, treatment with oxidized glutathione, paraquat, and ozone. Immunogold electron microscopy localized the hipI-SOD in the plasma membrane of sieve cells and the Golgi apparatus of albuminous cells. Moreover, high protein density was also detected in extracellular spaces such as secondary cell wall thickenings of the xylem and sclerenchyma and in intercellular spaces of parenchyma cells.
Yanhusuo (Corydalis yanhusuo W.T. Wang) is a well-known traditional Chinese medicine (TCM). In this study, we attempted to characterize in detail the signaling cascades that produce its anti-metastatic effect on the human breast cancer cell line, MDA-MB-231. We found that the yanhusuo extract inhibited the migration and invasion of MDA-MB-231 cells in vitro. In addition, the yanhusuo extract inhibited the mRNA expression and activity of metalloproteinase-9 (MMP-9). The anti-cancer metastasis effect of yanhusuo involved the activation of p38 and inhibition of ERK1/2 and SAPK/JNK mitogen-activated protein kinase (MAPK) signaling. Our experiments identified the biological activity of yanhusuo against cancer metastasis in vitro and provide a rationale for its further investigation.
Tuberization in potato ( Solanum tuberosum L.) is a developmental process that serves a double function, as a storage organ and as a vegetative propagation system. It is a multistep, complex process and the underlying mechanisms governing these overlapping steps are not fully understood. To understand the molecular basis of tuberization in potato, a comparative proteomic approach has been applied to monitor differentially expressed proteins at different development stages using two-dimensional gel electrophoresis (2-DE). The differentially displayed proteomes revealed 219 protein spots that change their intensities more than 2.5-fold. The LC-ES-MS/MS analyses led to the identification of 97 differentially regulated proteins that include predicted and novel tuber-specific proteins. Nonhierarchical clustering revealed coexpression patterns of functionally similar proteins. The expression of reactive oxygen species catabolizing enzymes, viz., superoxide dismutase, ascorbate peroxidase and catalase, were induced by more than 2-fold indicating their possible role during the developmental transition from stolons into tubers. We demonstrate that nearly 100 proteins, some presumably associated with tuber cell differentiation, regulate diverse functions like protein biogenesis and storage, bioenergy and metabolism, and cell defense and rescue impinge on the complexity of tuber development in potato.
Cytoplasmic aggregation is an early resistance-associated event that is observed in potato tissues either after penetration of an incompatible race of Phytophthora infestans, the potato late blight fungus, or after treatment with hyphal wall components (HWC) prepared from P. infestans. In potato cells in suspension culture, the number of cells with cytoplasmic aggregation increased upon treatment with HWC, but such an increase was suppressed by treatment with cytochalasin D prior to treatment with HWC. This result suggested that cytoplasmic aggregation in cultured potato cells might be connected with the association of actin filaments. To identify the molecular basis of cytoplasmic aggregation, we purified actin and actin-related proteins by affinity chromatography on a column of immobilized DNase I from cultured potato cells and isolated proteins of 43 kDa, 32 kDa and 22 kDa. Analysis of the amino-terminal amino acid sequences indicated that the 43 kDa, 32 kDa and 22 kDa proteins were potato actin, basic chitinase and osmotin-like protein, respectively. This conclusion was supported by the results of Western blotting analysis of the 43 kDa and 32 kDa proteins with antibodies against actin and basic chitinase. Binding analysis with actin coupled to actin-specific antibodies and biotinylated actin suggested that the 32 kDa and 22 kDa proteins had actin-binding activity. In addition, examination of biomolecular interactions using an optical biosensor confirmed the binding of chitinase to actin. These results imply the possibility that basic chitinase and osmotin-like protein might be involved in cytoplasmic aggregation, hereby participating. In the potato cell's defense against attack by pathogen.
Genomic sequencing has made it clear that a large fraction of the genes specifying the core biological functions are shared by all eukaryotes. Knowledge of the biological role of such shared proteins in one organism can often be transferred to other organisms. The goal of the Gene Ontology Consortium is to produce a dynamic, controlled vocabulary that can be applied to all eukaryotes even as knowledge of gene and protein roles in cells is accumulating and changing. To this end, three independent ontologies accessible on the World-Wide Web (http://www.geneontology.org) are being constructed: biological process, molecular function and cellular component.
The oxidative burst, the generation of reactive oxygen species (ROS) in response to microbial pathogen attack, is a ubiquitous early part of the resistance mechanisms of plant cells. It has also become apparent from the study of a number of plant-pathogen interactions and those modelled by elicitor treatment of cultured cells that there may be more than one mechanism operating. However, one mechanism may be dominant in any given species. NADPH oxidases have been implicated in a number of systems and have been cloned and characterized. However, the enzyme system which is the major source of ROS in French bean (Phaseolus vulgaris) cells treated with a cell wall elicitor from Colletotrichum lindemuthianum, appears to be dependent on an exocellular peroxidase. The second component, the extracellular alkalinization, occurs as a result of the Ca(2+) and proton influxes and the K(+) efflux common to most elicitation systems as one of the earliest responses. The third component, the actual reductant/substrate, has remained elusive. The low molecular weight compound composition of apoplastic fluid was compared before and after elicitation. The substrate only becomes available some min after elicitation and can be extracted, so that by comparing the profiles by LC-MS it has been possible to identify possible substrates. The mechanism has proved to be complex and may involve a number of low molecular weight components. Stimulation of H(2)O(2) production was observed with saturated fatty acids such as palmitate and stearate without concomitant oxylipin production. This biochemical evidence is supported by immunolocalization studies on papillae forming at bacterial infection sites that show the peroxidase isoform present at sites of H(2)O(2) production revealed by cerium chloride staining together with the cross-linked wall proteins and callose and callose synthase. The peroxidase has been cloned and expressed in Pichia pastoris and has been shown to catalyse the oxidation reaction with the same kinetics as the purified enzyme. Furthermore, Arabidopsis plants transformed heterologously using the French bean peroxidase in antisense orientation have proved to be highly susceptible to bacterial and fungal pathogens. Thus it is possible that Arabidopsis is another species with the potential to mount an apoplastic oxidative burst and these transformed plant lines may be useful to identify the peroxidase that is responsible.
Several algorithms have been described in the literature for protein identification by searching a sequence database using mass spectrometry data. In some approaches, the experimental data are peptide molecular weights from the digestion of a protein by an enzyme. Other approaches use tandem mass spectrometry (MS/MS) data from one or more peptides. Still others combine mass data with amino acid sequence data. We present results from a new computer program, Mascot, which integrates all three types of search. The scoring algorithm is probability based, which has a number of advantages: (i) A simple rule can be used to judge whether a result is significant or not. This is particularly useful in guarding against false positives. (ii) Scores can be com pared with those from other types of search, such as sequence homology. (iii) Search parameters can be readily optimised by iteration. The strengths and limitations of probability-based scoring are discussed, particularly in the context of high throughput, fully automated protein identification.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Flagellin, the main protein of the bacterial flagella, elicits defence responses and alters growth in Arabidopsis seedlings. Previously, we identified the FLS1 locus, which confers flagellin insensitivity in Ws-0. To identify additional components involved in flagellin perception, we screened for flagellin insensitivity mutants in the flagellin-sensitive accession La-er. Here, we describe the identification of a new locus, FLS2, by a map-based strategy. The FLS2 gene is ubiquitously expressed and encodes a putative receptor kinase. FLS2 shares structural and functional homologies with known plant resistance genes and with components involved in the innate immune system of mammals and insects.
In only a few perennial herbs is age determination of individual plants possible. The life cycle of Corydalis intermedia and C. solida is described. Plants have an age in years identical to the number of dead sheaths outside their underground tuber. Various ways to determine age in other species are reviewed: No. of dead layers around corm: Corydalis cava; no. of dead layers around tuber: Corydalis pumila, Drosera erythrorhiza, D. bulbosa; no. of dead remains of leaves/inflorescence stalks to the side of the corm: Philydrella pygmaea; no. of collaterals in corm: Liatris aspera; no. of nodes on rootstock: Trillium erectum; and no. of bands of high and low density of leaf scars on stems: Aeonium species.
The opium poppy (Papaver somniferum) belongs to the group of latex-containing plants. Latex is the milky-like fluid within laticifer cells. In this study, poppy latex was analyzed with respect to ultrastructure, alkaloid, and protein content. The main goal of this project was the examination of the proteins by two-dimensional gel electrophoresis. In a proteomics approach, we investigated two main fractions of the latex, namely the cytosolic serum and the sedimented fraction containing the alkaloid-accumulating vesicles. Of the serum, representing the protein-rich part of the latex, 75 spots were analyzed by internal peptide microsequencing, followed by a database searching. For 69 proteins a function could be assigned due to homology to known proteins, whereas six spots could not be identified. Furthermore, codeinone reductase, a representative of the specific enzyme system in morphine biosynthesis, could be detected within the cytosolic serum fraction. In the vesicle-containing pellet, 23 protein spots were analyzed. An attempt was also made to separate the vesicle pellet by density centrifugation, followed by investigation of the alkaloid content, ultrastructure, and protein pattern. This study describes the first database of soluble proteins present in the latex of P. somniferum
The tuber of potato (Solanum tuberosum) is commonly used as a model for underground storage organs. In this study, changes in the proteome were followed from tuberization, through tuber development and storage into the sprouting phase. Data interrogation using principal component analysis was able to clearly discriminate between the various stages of the tuber life cycle. Moreover, five well-defined protein expression patterns were found by hierarchical clustering. Altogether 150 proteins showing highly significant differences in abundance between specific stages in the life cycle were highlighted; 59 of these were identified. In addition, 50 proteins with smaller changes in abundance were identified, including several novel proteins. Most noticeably, the development process was characterized by the accumulation of the major storage protein patatin isoforms and enzymes involved in disease and defense reactions. Furthermore, enzymes involved in carbohydrate and energy metabolism and protein processing were associated with development but decreased during tuber maturation. These results represent the first comprehensive picture of many proteins involved in the tuber development and physiology.
A major problem hampering the use of MALDI-MS for quantitative measurements is the inhomogeneous distribution of analytes and matrices in sample preparations. In this study, an aerospray method was utilized for sample preparation method to improve sample homogeneity across stainless steel targets for quantitative analysis of quaternary ammonium alkaloids (QAAs). A selective precipitation reaction with Reinecke salt known to selectively trap QAAs was used to facilitate the separation and purification of QAAs from the complex crude plant extracts. Palmatine and berberine as the representative QAAs in commercial Rhizoma Corydalis were successfully quantified by introducing an internal standard with similar molecular properties as analytes. The LODs were found to be 0.07 fmol, for palmatine, and 0.24 fmol, respectively, for berberine. The content of QAAs of three commercial Rhizoma Corydalis was between 0.201 and 0.245% for palmatine, and 0.049–0.057% for berberine. Furthermore, MS/MS experiments based on the accurate-mass measurements were carried out by infrared multiphoton dissociation (IRMPD) for QAAs and the corresponding tertiary alkaloids, which offered additional selectivity for this quantitative analysis method. In the fragmentation of precursor ions from QAAs, only cleavage of substituted groups attached to the A- or D-ring was observed, while cleavage between B- and C-ring from tertiary alkaloids had occurred. This study offers a perspective into the utility of MALDI-FTMS as an alternate quantitative tool for QAAs, especially in complex plant extracts.
LEA (late embryogenesis abundant) proteins participate in plant stress tolerance responses, but the mechanisms by which protection occurs are not fully understood. In the present work the unfolded proteins from maize dry embryos were analyzed by mass spectrometry. Twenty embryo proteins were identified, and among them 13 corresponded to LEA-type proteins. We selected three major LEA proteins, Emb564, Rab17 and Mlg3, belonging to groups 1, 2 and 3, respectively, and we undertook a comparative study in order to highlight differences among them. The post-translational modifications of native proteins were analyzed and the anti-aggregation properties of recombinant Emb564, Rab17 and Mgl3 proteins were evaluated in vitro. In addition, the protective effects of the LEA proteins were assessed in living cells under stress in Escherichia coli cells and in Nicotiana bentamiana leaves agroinfiltrated with fluorescent LEA-green fluorescent protein (GFP) fusions. Protein visualization by confocal microscopy indicated that cells expressing Mg3-GFP showed reduced cell shrinkage effects during dehydration and that Rab17-GFP co-localized to leaf oil bodies after heat shock. Overall, the results highlight differences and suggest functional diversity among maize LEA groups.
Several algorithms have been described in the literature for protein identification by searching a sequence database using mass spectrometry data. In some approaches, the experimental data are peptide molecular weights from the digestion of a protein by an enzyme. Other approaches use tandem mass spectrometry (MS/MS) data from one or more peptides. Still others combine mass data with amino acid sequence data. We present results from a new computer program, Mascot, which integrates all three types of search. The scoring algorithm is probability based, which has a number of advantages: (i) A simple rule can be used to judge whether a result is significant or not. This is particularly useful in guarding against false positives. (ii) Scores can be compared with those from other types of search, such as sequence homology. (iii) Search parameters can be readily optimised by iteration. The strengths and limitations of probability-based scoring are discussed, particularly in the context of high throughput, fully automated protein identification.
The congruent development of computational technology, bioinformatics and analytical instrumentation makes proteomics ready for the next leap. Present-day state of the art proteomics grew from a descriptive method towards a full stake holder in systems biology. High throughput and genome wide studies are now made at the functional level. These include quantitative aspects, functional aspects with respect to protein interactions as well as post translational modifications and advanced computational methods that aid in predicting protein function and mapping these functionalities across the species border. In this review an overview is given of the current status of these aspects in plant studies with special attention to non-genomic model plants.
Mass spectrometry-based proteomics critically depends on algorithms for data interpretation. A current bottleneck in the rapid advance of proteomics technology is the closed nature and slow development cycle of vendor-supplied software solutions. We have created an open source software environment, called MSQuant, which allows visualization and validation of peptide identification results directly on the raw mass spectrometric data. MSQuant iteratively recalibrates MS data thereby significantly increasing mass accuracy leading to fewer false positive peptide identifications. Algorithms to increase data quality include an MS(3) score for peptide identification and a post-translational modification (PTM) score that determines the probability that a modification such as phosphorylation is placed at a specific residue in an identified peptide. MSQuant supports relative protein quantitation based on precursor ion intensities, including element labels (e.g., (15)N), residue labels (e.g., SILAC and ICAT), termini labels (e.g., (18)O), functional group labels (e.g., mTRAQ), and label-free ion intensity approaches. MSQuant is available, including an installer and supporting scripts, at http://msquant.sourceforge.net .
Corydalis turtschaninovii (CT) has been used for tumor therapy. However, it is still unclear how this herb prevents the diseases in experimental models. Nitric oxide (NO) as a potent macrophage-derived effector molecule against a variety of tumors has received increasing attention.
In this study, using mouse peritoneal macrophages, we have examined the mechanism by which CT regulates NO production.
When CT was used in combination with recombinant interferon-gamma (rIFN-gamma), there was a marked cooperative induction of NO production. However, CT had no effect on NO production by itself. The increase in NO synthesis was reflected as an increased amount of inducible NO synthase (iNOS) protein. The increased production of NO from rIFN-gamma plus CT-stimulated peritoneal macrophages was decreased by the treatment with N(G)-monomethyl-L-arginine or N(alpha)-Tosyl-Phe Chloromethyl Ketone, iNOS inhibitor. The increased production of NO from rIFN-gamma plus CT-stimulated cells was almost completely inhibited by pre-treatment with pyrrolidine dithiocarbamate, an inhibitor of nuclear factor kappa B (NF-kappaB). However, treatment of peritoneal macrophages with rIFN-gamma plus CT had no effect on the increase in tumor necrosis factor-alpha (TNF-alpha) production.
Our findings demonstrate that CT increases the production of NO and TNF-alpha by rIFN-gamma-primed macrophages and suggest that NF-kappaB plays a critical role in mediating these effects of CT.
Reactive oxygen species (ROS), such as the superoxide anion and hydrogen peroxide, are generated by the photosystems because photoexcited electrons are often generated in excess of requirements for CO2 fixation and used for reducing molecular oxygen, even under normal environmental conditions. Moreover, ROS generation is increased in chloroplasts if plants are subjected to stresses, such as drought, high salinity and chilling. Chloroplast-localized isoforms of ascorbate peroxidase and possibly peroxiredoxins assume the principal role of scavenging hydrogen peroxide. However, in vitro studies revealed that both types of peroxidases are easily damaged by hydrogen peroxide and lose their catalytic activities. This is one contributing factor for cellular damage that occurs under severe oxidative stress. In this review, I describe mechanisms of hydrogen peroxide-mediated inactivation of these two enzymes and discuss a reason why they became susceptible to damage by hydrogen peroxide.
Laticifers are specialized cells that occur in over 20 plant families in several unrelated angiosperm orders. Although laticifers are likely to be of polyphyletic origin, their occurrence is considered a morphological indicator of relatedness among species. The classification of laticifers is based on developmental patterns and overall morphology. The cytoplasmic latex exuded in response to damage often includes specialized metabolites, such as cardenolides, alkaloids and natural rubber. Laticifers provide an effective location to store defense metabolites, although not all latex-bearing plants accumulate bioactive natural products. Ecophysiological studies have shown that latex and its associated metabolites are vital for the defense of plants against insects. The anatomy, development and physiology of laticifers are discussed with a focus on evolutionary and ecological perspectives.
The novel classes of plant pathogenesis-related (PR) proteins identified during the last decade also include novel peptide families. This review specifically focuses on these pathogenesis-related peptides, including proteinase inhibitors (PR-6 family), plant defensins (PR-12 family), thionins (PR-13 family) and lipid transfer proteins (PR-14 family). For each family of PR peptides, the general features concerning occurrence, expression and possible functions of their members are described. Next, more specifically the occurrence of each PR peptide family in the model plant Arabidopsis thaliana is discussed. Single-gene studies performed on particular gene members of a PR peptide family are reported. In addition, expression data of yet undescribed gene members of that particular PR peptide family are presented by consultation of publicly available micro-array databases. Finally an update is provided on the potential role of these PR peptides in A. thaliana, with a focus on their possible involvement in plant defense.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
An improved procedure for staining of proteins following separation in polyacrylamide gels is described which utilizes the colloidal properties of Coomassie Brilliant Blue G-250 and R-250. The new method is based on addition of 20% v/v methanol and higher concentrations of ammonium sulfate to the staining solution previously described. The method combines the advantage of much shorter staining time with high sensitivity, a clear background not requiring destaining, stepwise staining, and stable fixation after staining. The method has been applied to staining of polyacrylamide gels after sodium dodecyl sulfate-electrophoresis and isoelectric focusing in carrier ampholyte-generated pH gradients.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
Chelidonium majus L. (Papaveraceae) has a long history as being useful for the treatment of many diseases in European countries. This plant is of great interest for its use also in Chinese herbal medicine. The plant contains, as major secondary metabolites, isoquinoline alkaloids, such as sanguinarine, chelidonine, chelerythrine, berberine and coptisine. Other compounds structurally unrelated to the alkaloids have been isolated from the aerial parts: several flavonoids and phenolic acids. C. majus extracts and its purified compounds exhibit interesting antiviral, antitumour and antimicrobial properties both in vitro and in vivo.
The flower-specific cDNA Tomf216 representing an mRNA encoding an osmotin-like protein was identified by the differential screening of a cDNA library produced from mRNA extracted from premeiotic tomato flowers (Lycopersicon esculentum). The cDNA is 894 nucleotides in length and encodes a protein of 252 amino acids. The deduced protein has a molecular mass of 27.2 kDa, an N-terminal region predicted to be a signal sequence, and a predicted molecular mass of 24.7 kDa for the mature protein. The protein contains 16 cysteine residues and has 30% to 32% amino acid sequence identity to pathogenesis-related osmotins. The gene is expressed in flowers and fruit with the highest levels of transcript accumulation in pistils from flowers at anthesis. No expression was detected in vegetative organs.
Bet v 1, the major birch pollen allergen, is a member of a multigene family; a number of isoforms and homologous proteins from closely related species (alder, hazel and hornbeam) has been isolated and their cDNAs cloned and characterized. Genomic clones coding for Bet v 1 and homologues from apple and hazel were isolated and sequenced. Some of these clones contained intervening sequences. The exon-intron formation is highly conserved throughout this family of pathogenesis-related proteins in dicot plants and is also found in Aopr1 (Asparagus officinalis), a monocol species. Phylogenetic analysis suggested a possible common origin of the intron position in these homologous proteins at codon 62 in various families of flowering plants, including Fagaceae, Rosaceae and Apiaceae. This conserved 'proto-splice site' may point to a structure/function relationship. A conserved sequence motif (P-loop) was also found in all members of this protein family. Moreover, there is a certain degree of sequence similarity among the proteins derived from various species throughout the dicots and the only monocot examined. This fact is reflected by cross-reactivity from monoclonal and polyclonal antibodies raised against Bet v 1.
Flagellin, the main protein of the bacterial flagella, elicits defence responses and alters growth in Arabidopsis seedlings. Previously, we identified the FLS1 locus, which confers flagellin insensitivity in Ws-0. To identify additional components involved in flagellin perception, we screened for flagellin insensitivity mutants in the flagellin-sensitive accession La-er. Here, we describe the identification of a new locus, FLS2, by a map-based strategy. The FLS2 gene is ubiquitously expressed and encodes a putative receptor kinase. FLS2 shares structural and functional homologies with known plant resistance genes and with components involved in the innate immune system of mammals and insects.
Osmotin is a tobacco PR-5 protein that has antifungal activity and is implicated in host-plant defense. We show here that osmotin induces apoptosis in Saccharomyces cerevisiae. Induction of apoptosis was correlated with intracellular accumulation of reactive oxygen species and was mediated by RAS2, but not RAS1. Osmotin treatment resulted in suppression of transcription of stress-responsive genes via the RAS2/cAMP pathway. It was therefore concluded that osmotin induced proapoptotic signaling in yeast. The results indicate that the ability of antimicrobial proteins to induce microbial apoptosis could be an important factor in determining a pathogen's virulence and could therefore be targeted for the design of new antifungal drugs.
Numerous sequences analogous to resistance (R) genes exist in plant genomes and could be involved in resistance traits. The aim of this study was to identify a large number of Brassica napus sequences related to R genes and also to test the adequacy of specific PCR-based tools for studying them. Different consensus primers were compared for their efficiency in amplifying resistance-gene analogues (RGAs) related to the nucleotide-binding-site subgroup of R genes. Specific primers were subsequently designed to fine-study the different RGAs and we tested their efficiency in three species related to B. napus: Brassica oleracea, Brassica rapa, and Arabidopsis thaliana. Forty-four B. napus RGAs were identified. Among 29 examined, at least one-third were expressed. Eighteen RGAs were mapped on 10 of the 19 B. napus linkage groups. The high variability within these sequences permitted discrimination of each genotype within a B. napus collection. The RGA-specific primers amplified RGAs in the B. oleracea and B. rapa genomes, but the sequences appear to be poorly conserved in A. thaliana. Specific RGA primers are a precise tool for studying known-sequence RGAs. These sequences represent interesting markers that could be correlated with resistance traits in B. napus or related Brassica genomes.
14-3-3 proteins were the first signaling molecules to be identified as discrete phosphoserine/threonine binding modules. This family of proteins, which includes seven isotypes in human cells and up to 15 in plants, plays critical roles in cell signaling events that control progress through the cell cycle, transcriptional alterations in response to environmental cues, and programmed cell death. Despite over 30 years of research, distinct roles for most isotypes remain unknown. Though 14-3-3 proteins perform different functions for different ligands, general mechanisms of 14-3-3 action include changes in activity of bound ligands, altered association of bound ligands with other cellular components, and changes in intracellular localization of 14-3-3-bound cargo. We present a speculative model where binding of 14-3-3 to multiple sites on some ligands results in global ligand conformational changes that mediate their biological effects. For these multi-site ligands, one binding site is likely to function as a 'gatekeeper' whose phosphorylation is necessary for 14-3-3 binding but may not always be sufficient for full biological activity. If correct, then 14-3-3 may prove to be a bona fide phosphodependent signaling chaperone.
The phloem is the major route for the transport of solutes and nutrients from source to sink organs in plants. The functional transport phloem consists of parenchymal tissue, enucleate sieve elements, and the intimately connected companion cells. The general absence of a nucleus and functional ribosomes in sieve tubes poses problems especially for damage avoidance and repair of sieve element components. To examine how sieve tubes can remain functional during oxidative stress, we analysed phloem sap of cucumber and pumpkin plants with respect to the presence of antioxidant defence enzymes, their enzymatic activity, and activity changes after exposure to drought stress. Using 1D SDS-PAGE and nano ESI MS/MS, the presence of proteins such as cytosolic Cu/Zn superoxide dismutase, monodehydroascorbate reductase, and peroxidase could be shown. Moreover, activities for several antioxidant enzymes (superoxide dismutase, dehydroascorbate reductase, peroxidase) in phloem exudate could be demonstrated. The activity of these enzymes in phloem sap from cucumber and pumpkin plants increased in response to drought stress. The presented results together with earlier findings provide evidence supporting the presence of a complete machinery of antioxidant defence enzymes and detoxifying metabolites important for avoiding damage to essential components of the sieve elements due to oxidative stress.