[Show abstract][Hide abstract] ABSTRACT: Fetal alcohol syndrome is a neurological and developmental disorder caused by exposure of developing brain to ethanol. Administration of osmotin to rat pups reduced ethanol-induced apoptosis in cortical and hippocampal neurons. Osmotin, a plant protein, mitigated the ethanol-induced increases in cytochrome c, cleaved caspase-3, and PARP-1. Osmotin and ethanol reduced ethanol neurotoxicity both in vivo and in vitro by reducing the protein levels of cleaved caspase-3, intracellular [Ca(2+)]cyt, and mitochondrial transmembrane potential collapse, and also upregulated antiapoptotic Bcl-2 protein. Osmotin is a homolog of adiponectin, and it controls energy metabolism via phosphorylation. Adiponectin can protect hippocampal neurons against ethanol-induced apoptosis. Abrogation of signaling via receptors AdipoR1 or AdipoR2, by transfection with siRNAs, reduced the ability of osmotin and adiponectin to protect neurons against ethanol-induced neurodegeneration. Metformin, an activator of AMPK (adenosine monophosphate-activated protein kinase), increased whereas Compound C, an inhibitor of AMPK pathway, reduced the ability of osmotin and adiponectin to protect against ethanol-induced apoptosis. Osmotin exerted its neuroprotection via Bcl-2 family proteins and activation of AMPK signaling pathway. Modulation of AMPK pathways by osmotin, adiponectin, and metformin hold promise as a preventive therapy for fetal alcohol syndrome.
Cell Death & Disease 03/2014; 5:e1150. · 5.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glyphosate is a widely applied broad-spectrum systemic herbicide that competitively inhibits the penultimate enzyme, 5-Enolpyruvylshikimate 3-Phosphate Synthase (EPSPS), of the shikimate pathway thereby causing deleterious effects. A glyphosate resistant Arabidopsis mutant (gre1) was isolated and genetic analyses indicated a dysfunctional red (R) and far-red (FR) light receptor, phytochrome B (phyB), causing this phenotype. This is consistent with increased glyphosate sensitivity and glyphosate-induced shikimate accumulation in low R:FR light, and the induction of genes encoding enzymes of the shikimate pathway in high R:FR light. Expression of the shikimate pathway genes exhibits diurnal oscillation and this is altered in the phyB mutant. Furthermore, transcript analysis suggests that this diurnal oscillation is not only dependent on phyB but also due to circadian regulatory mechanisms. Our data offer an explanation of the well-documented observation that glyphosate treatment at various times throughout the day, with their specific composition of light quality and intensity, results in different efficiencies of the herbicide.This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Adiponectin is a mammalian hormone that exerts anti-diabetic, anti-cancer and cardioprotective effects through interaction with its major ubiquitously expressed plasma membrane localized receptors, AdipoR1 and AdipoR2. Here, we report a Saccharomyces cerevisiae based method for investigating agonist-AdipoR interactions that is amenable for high-throughput scale-up and can be used to study both AdipoRs separately. Agonist-AdipoR1 interactions are detected using a split firefly luciferase assay based on reconstitution of firefly luciferase (Luc) activity due to juxtaposition of its N- and C-terminal fragments, NLuc and CLuc, by ligand induced interaction of the chimeric proteins CLuc-AdipoR1 and APPL1-NLuc (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif 1-NLuc) in a S. cerevisiae strain lacking the yeast homolog of AdipoRs (Izh2p). The assay monitors the earliest known step in the adiponectin-AdipoR anti-diabetic signaling cascade. We demonstrate that reconstituted Luc activity can be detected in colonies or cells using a CCD camera and quantified in cell suspensions using a microplate reader. AdipoR1-APPL1 interaction occurs in absence of ligand but can be stimulated specifically by agonists such as adiponectin and the tobacco protein osmotin that was shown to have AdipoR-dependent adiponectin-like biological activity in mammalian cells. To further validate this assay, we have modeled the three dimensional structures of receptor-ligand complexes of membrane-embedded AdipoR1 with cyclic peptides derived from osmotin or osmotin-like plant proteins. We demonstrate that the calculated AdipoR1-peptide binding energies correlate with the peptides' ability to behave as AdipoR1 agonists in the split luciferase assay. Further, we demonstrate agonist-AdipoR dependent activation of protein kinase A (PKA) signaling and AMP activated protein kinase (AMPK) phosphorylation in S. cerevisiae, which are homologous to important mammalian adiponectin-AdipoR1 signaling pathways. This system should facilitate the development of therapeutic inventions targeting adiponectin and/or AdipoR physiology.
PLoS ONE 06/2013; 8(6):e65454. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Arabidopsis thaliana YUCCA family of flavin monooxygenase proteins catalyses a rate-limiting step in de novo auxin biosynthesis. A YUCCA6 activation mutant, yuc6-1D, has been shown to contain an elevated free IAA level and to display typical high-auxin phenotypes. It is reported here that Arabidopsis plants over-expressing YUCCA6, such as the yuc6-1D activation mutant and 35S:YUC6 transgenic plants, displayed dramatic longevity. In addition, plants over-expressing YUCCA6 exhibited classical, delayed dark-induced and hormone-induced senescence in assays using detached rosette leaves. However, plants over-expressing an allele of YUCCA6, that carries mutations in the NADPH cofactor binding site, exhibited neither delayed leaf senescence phenotypes nor phenotypes typical of auxin overproduction. When the level of free IAA was reduced in yuc6-1D by conjugation to lysine, yuc6-1D leaves senesced at a rate similar to the wild-type leaves. Dark-induced senescence in detached leaves was accompanied by a decrease in their free IAA content, by the reduced expression of auxin biosynthesis enzymes such as YUCCA1 and YUCCA6 that increase cellular free IAA levels, and by the increased expression of auxin-conjugating enzymes encoded by the GH3 genes that reduce the cellular free auxin levels. Reduced transcript abundances of SAG12, NAC1, and NAC6 during senescence in yuc6-1D compared with the wild type suggested that auxin delays senescence by directly or indirectly regulating the expression of senescence-associated genes.
Journal of Experimental Botany 04/2011; 62(11):3981-92. · 5.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hypoadiponectinemia has been associated with states of chronic inflammation in humans. Mesenteric fat hypertrophy and low adiponectin have been described in patients with Crohn's disease. We investigated whether adiponectin and the plant-derived homolog, osmotin, are beneficial in a murine model of colitis.
C57BL/6 mice were injected (i.v.) with an adenoviral construct encoding the full-length murine adiponectin gene (AN+DSS) or a reporter-LacZ (Ctr and V+DSS groups) prior to DSS colitis protocol. In another experiment, mice with DSS colitis received either osmotin (Osm+DSS) or saline (DSS) via osmotic pumps. Disease progression and severity were evaluated using body weight, stool consistency, rectal bleeding, colon lengths, and histology. In vitro experiments were carried out in bone marrow-derived dendritic cells.
Mice overexpressing adiponectin had lower expression of proinflammatory cytokines (TNF, IL-1β), adipokines (angiotensin, osteopontin), and cellular stress and apoptosis markers. These mice had higher levels of IL-10, alternative macrophage marker, arginase 1, and leukoprotease inhibitor. The plant adiponectin homolog osmotin similarly improved colitis outcome and induced robust IL-10 secretion. LPS induced a state of adiponectin resistance in dendritic cells that was reversed by treatment with PPARγ agonist and retinoic acid.
Adiponectin exerted protective effects during murine DSS colitis. It had a broad activity that encompassed cytokines, chemotactic factors as well as processes that assure cell viability during stressful conditions. Reducing adiponectin resistance or using plant-derived adiponectin homologs may become therapeutic options in inflammatory bowel disease.
Digestive Diseases and Sciences 04/2011; 56(10):2818-32. · 2.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fusarium oxysporum is the causative agent of fungal wilt disease in a variety of crops. The capacity of a fungal pathogen such as F. oxysporum f. sp. nicotianae to establish infection on its tobacco (Nicotiana tabacum) host depends in part on its capacity to evade the toxicity of tobacco defense proteins, such as osmotin. Fusarium genes that control resistance to osmotin would therefore reflect coevolutionary pressures and include genes that control mutual recognition, avoidance, and detoxification. We identified FOR (Fusarium Osmotin Resistance) genes on the basis of their ability to confer osmotin resistance to an osmotin-sensitive strain of Saccharomyces cerevisiae. FOR1 encodes a putative cell wall glycoprotein. FOR2 encodes the structural gene for glutamine:fructose-6-phosphate amidotransferase, the first and rate-limiting step in the biosynthesis of hexosamine and cell wall chitin. FOR3 encodes a homolog of SSD1, which controls cell wall composition, longevity, and virulence in S. cerevisiae. A for3 null mutation increased osmotin sensitivity of conidia and hyphae of F. oxysporum f. sp. nicotianae and also reduced cell wall beta-1,3-glucan content. Together our findings show that conserved fungal genes that determine cell wall properties play a crucial role in regulating fungal susceptibility to the plant defense protein osmotin.
[Show abstract][Hide abstract] ABSTRACT: Innate immunity in plants is manifested by a complex array of antimicrobial processes that includes induction of sets of pathogenesis-related (PR) proteins. The availability of genomic data has made clear that each PR-protein family in a species is represented by several genes. Microarray data in public databases show that in most families, including the PR-5 family surveyed here, the expression of only few family members is defense associated. Genetic studies show that depending on their nutrient acquisition strategy, pathogens induce distinct but overlapping sets of PR genes, suggesting a connection to energy or resource allocation. PR-5 proteins have a clearly recognizable structure that is referred to as the thaumatin (THN) domain, which can be overlapped with mammalian Complement 1q-tumor necrosis factor (C1q-TNF) domains such as that of the mammalian hormone adiponectin. The occurrence of THN domain proteins is widespread. Similarities between THN domain proteins and mammalian C1q-TNF family proteins include their ligands and their subcellular locations. Osmotin (tobacco PR-5c) regulates energy balance signaling in mammalian cells by interaction with adiponectin receptors by a pathway that shares components with plant energy and stress signaling pathways. These data suggest additional roles for PR-5 proteins, as scaffolds and/or in signaling, particularly in regulating energy balance.
[Show abstract][Hide abstract] ABSTRACT: Heat-stable mycelial extracts of the nonpathogenic fungus Trichoderma longibrachiatum induced resistance in tobacco seedlings (Nicotiana tabacum L. cv. Wisconsin 38) to the pathogen Phytophthora parasitica var. nicotianae (race 0), which did not involve a hypersensitive response. Resistance could not be induced with mycelial extract prepared in the same manner from P. parasitica. The nonpathogenic mycelial extract induced expression of PR-1b and osmotin (PR-5) genes to a higher level than did mycelial extract from the pathogenic fungus. The tissue-specific pattern of PR gene induction by the nonpathogenic mycelial extract was different from that of the pathogenic mycelial extract and was consistent with the ability of the former to cause disease resistance. The expression patterns of these two PR genes and the accumulations of their encoded proteins also were affected by salicylic acid (SA), methyl jasmonate (MeJA), ethylene (E) and combinations of these plant signal messengers. However, only combined SA and MeJA treatment mimicked the pattern of PR gene mRNA and protein accumulation induced by the nonpathogenic mycelial extract. E inhibitors blocked both mycelial extract-induced and SA/MeJA-induced PR gene expression, and the cis pattern of responsiveness on the osmotin promoter was the same for the mycelial extract, SA, E, or E/MeJA. Seedlings treated with P. parasitica spores in the presence of SA/MeJA were protected from pathogen colonization. However, these seedlings exhibited symptoms of cell death (disease symptoms) both in the absence and presence of P. parasitica spores, in contrast to seedlings treated with nonpathogenic mycelial extract, which remained healthy. These results suggest that the signal transduction pathways for elicitation of defense responses by exogenously applied heat-stable nonpathogenic mycelial extract and SA/MeJA overlap at the point of PR protein induction but are not identical.
Physiologia Plantarum 04/2006; 100(2):341 - 352. · 3.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: ABSTRACTA cDNA clone encoding an isoform of the plasma membrane H+-ATPase was isolated from Nicotiana tabacum. The steady-state plasma membrane H+-ATPase message levels were the same in unadapted tobacco cells and tobacco cells adapted to 428 mol m−3 NaCl. When cells adapted to 428 mol m−3 NaCl maintained in the absence of NaCl (deadapted) for an excess of 100 passages were exposed to 400 mol m−3 NaCl for 24 h, there was an increased accumulation of plasma membrane H+-ATPase message. The NaCl responsiveness of the deadapted cells was dependent upon the growth cycle stage. Alterations in the levels of plasma membrane FT-ATPase message during the growth cycle support a role for the H+-ATPase in cell growth. These results document the induction by NaCl of plasma membrane FT-ATPase message accumulation in tobacco cells, and suggest that enhanced expression of the plasma membrane FT-ATPase has a role in the short term response of cells of NaCl, but is not necessarily involved in long-term adaptation.
[Show abstract][Hide abstract] ABSTRACT: Cultured tobacco cells (Nicotiana tabacum L. cv. Wisconsin 38) adapted to NaCl exhibited significant morphological and physiological changes. Adapted cells remained smaller and more isodiametric at maturity than unadapted cells. The vacuole increased in size relative to the cytoplasm and an extensive network of transvacuolar membrane strands developed. These changes altered the surface contact area between the cytoplasm and the vacuole substantially. In addition, the network of Hechtian strands that anchor the cortical structure to the cell wall became more extensively branched possibly facilitating surface contact of the cytoplasm to the extracellular matrix. Many changes in membrane proteins could also be identified after NaCl adaptation. In particular, a 50-kDa protein that is associated with the plasma membrane and tonoplast was induced during adaptation. Immunocytochemical localization indicated that this 50-kDa protein is associated with Golgi vesicles. By immunoscreening using anti-50-kDa antibody, a 1.71-kb cDNA clone (p50C) was isolated from a λ-ZAP cDNA expression library. The sequence of p50C did not show any significant identity with other genes. Because of the very low abundance of the p50C message, quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze p50C gene expression. Immunoblot and quantitative RT-PCR analyses indicated that the expression of this gene is regulated developmentally since the mRNA and protein increased with age in salt-adapted cells but decreased with age in unadapted cells. Also in tobacco plants, p50C mRNA was more abundant in younger leaves than in older leaves. The gene was responsive to NaCl in tobacco cells and to ABA in tobacco seedlings.
Physiologia Plantarum 04/2006; 98(3):505 - 516. · 3.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Adaptive silver films (ASFs) have been studied as a substrate for protein microarrays. Vacuum evaporated silver films fabricated at certain range of evaporation parameters allow fine rearrangement of the silver nanostructure under protein depositions in buffer solution. Proteins restructure and stabilize the ASF to increase the surface-enhanced Raman scattering (SERS) signal from a monolayer of molecules. Preliminary evidence indicates that the adaptive property of the substrates make them appropriate for protein microarray assays. Head-to-head comparisons with two commercial substrates have been performed. Protein binding was quantified on the microarray using the streptavidinCy3/biotinylated goat IgG protein pair. With fluorescence detection, the performance of ASF substrates was comparable with SuperAldehyde and SuperEpoxy substrates. Additionally, the ASF is also a SERS substrate and this provides an additional tool for analysis. It is found that the SERS spectra of the streptavidinCy5 fluorescence reporter bound to true and bound to false sites show distinct difference.
[Show abstract][Hide abstract] ABSTRACT: The antifungal activity of the PR-5 family of plant defense proteins has been suspected to involve specific plasma membrane component(s) of the fungal target. Osmotin is a tobacco PR-5 family protein that induces apoptosis in the yeast Saccharomyces cerevisiae. We show here that the protein encoded by ORE20/PHO36 (YOL002c), a seven transmembrane domain receptor-like polypeptide that regulates lipid and phosphate metabolism, is an osmotin binding plasma membrane protein that is required for full sensitivity to osmotin. PHO36 functions upstream of RAS2 in the osmotin-induced apoptotic pathway. The mammalian homolog of PHO36 is a receptor for the hormone adiponectin and regulates cellular lipid and sugar metabolism. Osmotin and adiponectin, the corresponding "receptor" binding proteins, do not share sequence similarity. However, the beta barrel domain of both proteins can be overlapped, and osmotin, like adiponectin, activates AMP kinase in C2C12 myocytes via adiponectin receptors.
[Show abstract][Hide abstract] ABSTRACT: Fusarium oxysporum f. sp. nicotianae is a causal agent for vascular wilt disease in tobacco. It is sensitive to osmotin, a tobacco pathogenesis-related protein (PR-5) that is implicated in plant defense against phytopathogenic fungi. We show that osmotin susceptibility of F. oxysporum f. sp. nicotianae was reduced by overexpression of the heterologous cell wall glycoprotein Saccharomyces cerevisiae protein containing inverted repeats (PIR2), a member of the PIR family of fungal cell wall glycoproteins that protect S. cerevisiae from the toxic action of osmotin. S. cerevisiae PIR2 was targeted to the cell wall of F. oxysporum. Disease severity and fungal growth were increased in tobacco seedlings inoculated with F. oxysporum transformed with PIR2 compared to seedlings infected with untransformed F. oxysporum or that transformed with vector, although accumulation of transcript and protein of defense genes was similar. The results show that fungal cell wall components can increase resistance to plant defense proteins and affect virulence.
The Plant Journal 12/2003; 36(3):390-400. · 6.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Verticillium dahliae Klebahn is a soil-borne fungal pathogen causing vascular diseases. The pathogen penetrates the host through the roots, spreads through the xylem, and systemically colonizes both resistant and susceptible genotypes. To elucidate the genetic and molecular bases of plant-Verticillium interactions, we have developed a pathosystem utilizing Arabidopsis thaliana and an isolate of V. dahliae pathogenic to both cruciferous and non-cruciferous crops. Relative tolerance (based on symptom severity) but no immunity was found in a survey of Arabidopsis ecotypes. Anthocyanin accumulation, stunting, and chlorosis were common symptoms. Specific responses of the more susceptible ecotype Columbia were induction of early flowering and dying. The more tolerant ecotype C-24 was characterized by pathogen-induced delay of transition to flowering and mild chlorosis symptoms. Genetic analysis indicated that a single dominant locus, Verticillium dahliae-tolerance (VET1), likely functioning also as a negative regulator of the transition to flowering, was able to convey increased tolerance. VET1 was mapped on chromosome IV. The differential symptom responses observed between ecotypes were not correlated with different rates of fungal tissue colonization or with differential transcript accumulation of PR-1 and PDF1.2 defense genes whose activation was not detected during the Arabidopsis-V. dahliae interaction. Impairment in salicylic acid (SA)- or jasmonic acid (JA)-dependent signaling did not cause hypersensitivity to the fungal infection, whereas ethylene insensitivity led to reduced chlorosis and ABA deficiency to reduced anthocyanin accumulation. The results of this study clearly indicated that the ability of V. dahliae to induce disease symptoms is also connected to the genetic control of development and life span in Arabidopsis.
The Plant Journal 10/2003; 35(5):574-87. · 6.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Plants are major targets of microbes seeking a source of nutrition. A complex array of interactions between plants and microbes has evolved that re- flects both the nutrient acquisition strategies of mi- crobes and defense strategies of plants. Part of plant defense strategy includes an active offense against invading microbes using an array of antimicrobial gene products. Within the context of the overall plant-microbe interaction, we attempt here to empha- size the role of antimicrobial proteins (typically, over 100 amino acid residues) and peptides (typically, 30-60 amino acid residues) in plant defense. The majority of plant-microbe encounters do not result in disease. Preformed factors including consti- tutively expressed waxes, cell wall components, anti- microbial peptides, proteins, and non-proteinaceous secondary metabolites that deter invasion have been proposed to contribute significantly to the host range of pathogens (Garcia-Olmedo et al., 1998; Morrisey and Osbourn, 1999; Heath, 2000). The importance of preformed defenses has been inferred from the obser- vation that plants can be rendered susceptible by a deficiency in the production of these secondary me- tabolites or by the abilities of pathogens to degrade them (Morrisey and Osbourn, 1999; Papadopoulou et al., 1999).
[Show abstract][Hide abstract] ABSTRACT: SummaryA cDNA clone pCZ1, with a 1.1 kb insert, was isolated from a NaCI-adapted tobacco cell cDNA library that encodes an apparently full-length 29 kDa protein (251 amino acids) with a calculated pl of 5.7. The encoded peptide had a high amino acid sequence identity with bovine 14-3-3 protein which was originally found as an abundant protein in the animal central nervous system. Recently, proteins with sequence identity to 14-3-3 protein have also been found in plants, insects and yeast, and appear to have diverse physiological functions. Similar to the bovine brain 14-3-3 protein, the recombinant pCZ1 protein stimulated ADP-ribosylation of protein substrate by ADP-ribosyl-transferase from the plant and animal pathogenic bacterium Pseudomonas aeruginosa. This recombinant protein also inhibited protein kinase C activity in vitro. Southern blot analyses indicated that most likely five genes encoding 14-3-3-like proteins are present in tobacco. The pCZ1 cDNA insert hybridized to a single mRNA of 1.1 kb from cultured tobacco cells. The level of this mRNA transcript in tobacco cells was downregulated upon adaptation to NaCl but was unaffected by short-term treatment with NaCl, ABA or ethylene. In tobacco plants, expression of transcript that hybridized to pCZ1 was tissue specific, and was most abundant in roots and flower parts. Monoclonal antibody raised against GF14 protein, a maize protein with substantial sequence identity with 14-3-3 protein detected two bands on SDS-PAGE of total proteins from unadapted tobacco cells and only a single band from cells adapted to NaCl. The GF14 antibody was also used to illustrate that the G-box element of a salt-induced gene is associated with a 14-3-3-type protein.
[Show abstract][Hide abstract] ABSTRACT: An interesting observation, reported for transgenic plants that have been engineered to overproduce osmolytes, is that they often exhibit impaired growth in the absence of stress. As growth reduction and accumulation of osmolytes both typically result from adaptation, we hypothesized that growth reduction may actually result from osmolyte accumulation. To examine this possibility more closely, intracellular proline level was manipulated by expressing mutated derivatives of tomPRO2 (a Delta(1)-pyrroline-5-carboxylate synthetase, P5CS, from tomato) in Saccharomyces cerevisiae. This was done in the presence and absence of a functional proline oxidase, followed by selection and screening for increased accumulation of proline in the absence of any stress. Here we show, in support of our hypothesis, that the level of proline accumulation and the amount of growth are inversely correlated in cells grown under normal osmotic conditions. In addition, the intracellular concentration of proline also resulted in increases in ploidy level, vacuolation and altered accumulation of several different transcripts related to cell division and gene expression control. Because these cellular modifications are common responses to salt stress in both yeast and plants, we propose that proline and other osmolytes may act as a signaling/regulatory molecule able to activate multiple responses that are part of the adaptation process. As in previous studies with transgenic plants that overaccumulate osmolytes, we observed some increase in relative growth of proline-overaccumulating cells in mild hyperosmotic stress.
The Plant Journal 10/2002; 31(6):699-712. · 6.82 Impact Factor