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The newly defined phytohormones strigolactones (SLs) were recently shown to act as regulators of root development. Their positive effect on root-hair (RH) elongation enabled examination of their cross talk with auxin and ethylene. Analysis of wild-type plants and hormone-signaling mutants combined with hormonal treatments suggested that SLs and ethylene regulate RH elongation via a common regulatory pathway, in which ethylene is epistatic to SLs. The SL and auxin hormonal pathways were suggested to converge for regulation of RH elongation; this convergence was suggested to be mediated via the ethylene pathway, and to include regulation of auxin transport.Key words: strigolactone, auxin, ethylene, root, root hair, lateral rootStrigolactones (SLs) are newly identified phytohormones that act as long-distance shoot-branching inhibitors (reviewed in ref. 1). In Arabidopsis, SLs have been shown to be regulators of root development and architecture, by modulating primary root elongation and lateral root formation.2,3 In addition, they were shown to have a positive effect on root-hair (RH) elongation.2 All of these effects are mediated via the MAX2 F-box.2,3In addition to SLs, two other plant hormones, auxin and ethylene, have been shown to affect root development, including lateral root formation and RH elongation.46 Since all three phytohormones (SLs, auxin and ethylene) were shown to have a positive effect on RH elongation, we examined the epistatic relations between them by examining RH length.7 Our results led to the conclusion that SLs and ethylene are in the same pathway regulating RH elongation, where ethylene may be epistatic to SLs.7 Moreover, auxin signaling was shown to be needed to some extent for the RH response to SLs: the auxin-insensitive mutant tir1-1,8 was less sensitive to SLs than the wild type under low SL concentrations.7On the one hand, ethylene has been shown to induce the auxin response,912 auxin synthesis in the root apex,11,12 and acropetal and basipetal auxin transport in the root.4,13 On the other, ethylene has been shown to be epistatic to SLs in the SL-induced RH-elongation response.7 Therefore, it might be that at least for RH elongation, SLs are in direct cross talk with ethylene, whereas the cross talk between SL and auxin pathways may converge through that of ethylene.7 The reduced response to SLs in tir1-1 may be derived from its reduced ethylene sensitivity;7,14 this is in line with the notion of the ethylene pathway being a mediator in the cross talk between the SL and auxin pathways.The suggested ethylene-mediated convergence of auxin and SLs may be extended also to lateral root formation, and may involve regulation of auxin transport. In the root, SLs have been suggested to affect auxin efflux,3,15 whereas ethylene has been shown to have a positive effect on auxin transport.4,13 Hence, it might be that in the root, the SLs'' effect on auxin flux is mediated, at least in part, via the ethylene pathway. Ethylene''s ability to increase auxin transport in roots was associated with its negative effect on lateral root formation: ethylene was suggested to enhance polar IAA transport, leading to alterations in the quantity of auxin that unloads into the tissues to drive lateral root formation.4 Under conditions of sufficient phosphate, SL''s effect was similar to that of ethylene: SLs reduced the appearance of lateral roots; this was explained by their ability to change auxin flux.3 Taken together, one possibility is that the SLs'' ability to affect auxin flux and thereby lateral root formation in the roots is mediated by induction of ethylene synthesis.To conclude, root development may be regulated by a network of auxin, SL and ethylene cross talk.7 The possibility that similar networks exist elsewhere in the SLs'' regulation of plant development, including shoot architecture, cannot be excluded.  相似文献   

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Shoot elongation is a vital process for plant development and productivity, in both ecological and economic contexts. Auxin and bioactive gibberellins (GAs), such as GA1, play critical roles in the control of elongation,13 along with environmental and endogenous factors, including other hormones such as the brassinosteroids.4,5 The effect of auxins, such as indole-3-acetic acid (IAA), is at least in part mediated by its effect on GA metabolism,6 since auxin upregulates biosynthesis genes such as GA 3-oxidase and GA 20-oxidase and downregulates GA catabolism genes such as GA 2-oxidases, leading to elevated levels of bioactive GA1.7 In our recent paper,1 we have provided evidence that this action of IAA is largely independent of DELLA proteins, the negative regulators of GA action,8,9 since the auxin effects are still present in the DELLA-deficient la cry-s genotype of pea. This was a crucial issue to resolve, since like auxin, the DELLAs also promote GA1 synthesis and inhibit its deactivation. DELLAs are deactivated by GA, and thereby mediate a feedback system by which bioactive GA regulates its own level.10 However, our recent results,1 in themselves, do not show the generality of the auxin-GA relationship across species and phylogenetic groups or across different tissue types and responses. Further, they do not touch on the ecological benefits of the auxin-GA interaction. These issues are discussed below as well as the need for the development of suitable experimental systems to allow this process to be examined.Key words: auxin, gibberellins, DELLA proteins, interactions, elongation  相似文献   

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In young Arabidopsis seedlings, retrograde signaling from plastids regulates the expression of photosynthesis-associated nuclear genes in response to the developmental and functional state of the chloroplasts. The chloroplast-located PPR protein GUN1 is required for signalling following disruption of plastid protein synthesis early in seedling development before full photosynthetic competence has been achieved. Recently we showed that sucrose repression and the correct temporal expression of LHCB1, encoding a light-harvesting chlorophyll protein associated with photosystem II, are perturbed in gun1 mutant seedlings.1 Additionally, we demonstrated that in gun1 seedlings anthocyanin accumulation and the expression of the “early” anthocyanin-biosynthesis genes is perturbed. Early seedling development, predominantly at the stage of hypocotyl elongation and cotyledon expansion, is also affected in gun1 seedlings in response to sucrose, ABA and disruption of plastid protein synthesis by lincomycin. These findings indicate a central role for GUN1 in plastid, sucrose and ABA signalling in early seedling development.Key words: ABA, ABI4, anthocyanin, chloroplast, GUN1, retrograde signalling, sucroseArabidopsis seedlings develop in response to light and other environmental cues. In young seedlings, development is fuelled by mobilization of lipid reserves until chloroplast biogenesis is complete and the seedlings can make the transition to phototrophic growth. The majority of proteins with functions related to photosynthesis are encoded by the nuclear genome, and their expression is coordinated with the expression of genes in the chloroplast genome. In developing seedlings, retrograde signaling from chloroplasts to the nucleus regulates the expression of these nuclear genes and is dependent on the developmental and functional status of the chloroplast. Two classes of gun (genomes uncoupled) mutants defective in retrograde signalling have been identified in Arabidopsis: the first, which comprises gun2–gun5, involves mutations in genes encoding components of tetrapyrrole biosynthesis.2,3 The other comprises gun1, which has mutations in a nuclear gene encoding a plastid-located pentatricopeptide repeat (PPR) protein with an SMR (small MutS-related) domain near the C-terminus.4,5 PPR proteins are known to have roles in RNA processing6 and the SMR domain of GUN1 has been shown to bind DNA,4 but the specific functions of these domains in GUN1 are not yet established. However, GUN1 has been shown to be involved in plastid gene expression-dependent,7 redox,4 ABA1,4 and sucrose signaling,1,4,8 as well as light quality and intensity sensing pathways.911 In addition, GUN1 has been shown to influence anthocyanin biosynthesis, hypocotyl extension and cotyledon expansion.1,11  相似文献   

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Auxin is a phytohormone essential for plant development. Due to the high redundancy in auxin biosynthesis, the role of auxin biosynthesis in embryogenesis and seedling development, vascular and flower development, shade avoidance and ethylene response were revealed only recently. We previously reported that a vitamin B6 biosynthesis mutant pdx1 exhibits a short-root phenotype with reduced meristematic zone and short mature cells. By reciprocal grafting, we now have found that the pdx1 short root is caused by a root locally generated signal. The mutant root tips are defective in callus induction and have reduced DR5::GUS activity, but maintain relatively normal auxin response. Genetic analysis indicates that pdx1 mutant could suppress the root hair and root growth phenotypes of the auxin overproduction mutant yucca on medium supplemented with tryptophan (Trp), suggesting that the conversion from Trp to auxin is impaired in pdx1 roots. Here we present data showing that pdx1 mutant is more tolerant to 5-methyl anthranilate, an analogue of the Trp biosynthetic intermediate anthranilate, demonstrating that pdx1 is also defective in the conversion from anthranilate to auxin precursor tryptophan. Our data suggest that locally synthesized auxin may play an important role in the postembryonic root growth.Key words: auxin synthesis, root, PLP, PDX1The plant hormone auxin modulates many aspects of growth and development including cell division and cell expansion, leaf initiation, root development, embryo and fruit development, pattern formation, tropism, apical dominance and vascular tissue differentiation.13 Indole-3-acetic acid (IAA) is the major naturally occurring auxin. IAA can be synthesized in cotyledons, leaves and roots, with young developing leaves having the highest capacity.4,5Auxin most often acts in tissues or cells remote from its synthetic sites, and thus depends on non-polar phloem transport as well as a highly regulated intercellular polar transport system for its distribution.2The importance of local auxin biosynthesis in plant growth and development has been masked by observations that impaired long-distance auxin transport can result in severe growth or developmental defects.3,6 Furthermore, a few mutants with reduced free IAA contents display phenotypes similar to those caused by impaired long-distance auxin transport. These phenotypes include defective vascular tissues and flower development, short primary roots and reduced apical dominance, or impaired shade avoidance and ethylene response.715 Since these phenotypes most often could not be rescued by exogenous auxin application, it is difficult to attribute such defects to altered local auxin biosynthesis. By complementing double, triple or quadruple mutants of four Arabidopsis shoot-abundant auxin biosynthesis YUCCA genes with specific YUCCA promoters driven bacterial auxin biosynthesis iaaM gene, Cheng et al. provided unambiguous evidence that auxin biosynthesis is indispensable for embryo, flower and vascular tissue development.8,13 Importantly, it is clear that auxin synthesized by YUCCAs is not functionally interchangeable among different organs, supporting the notion that auxin synthesized by YUCCAs mainly functions locally or in a short range.6,8,13The central role of auxin in root meristem patterning and maintenance is well documented,1,2,16 but the source of such IAA is still unclear. When 14C-labeled IAA was applied to the five-day-old pea apical bud, the radioactivity could be detected in lateral root primordia but not the apical region of primary roots.17 Moreover, removal of the shoot only slightly affected elongation of the primary root, and localized application of auxin polar transport inhibitor naphthylphthalamic acid (NPA) at the primary root tip exerted more profound inhibitory effect on root elongation than at any other site.18 These results suggest that auxin generated near the root tip may play a more important role in primary root growth than that transported from the shoot. In line with this notion, Arabidopsis roots have been shown to harbor multiple auxin biosynthesis sites including root tips and the region upward from the tip.4Many steps of tryptophan synthesis and its conversion to auxin involve transamination reactions, which require the vitamin B6 pyridoxal 5-phosphate (PLP) as a cofactor. We previously reported that the Arabidopsis mutant pdx1 that is defective in vitamin B6 biosynthesis displays dramatically reduced primary root growth with smaller meristematic zone and shorter mature cortical cells.19 In the current investigation, we found that the root tips of pdx1 have reduced cell division capability and reduced DR5::GUS activity, although the induction of this reporter gene by exogenous auxin was not changed. Reciprocal grafting indicates that the short-root phenotype of pdx1 is caused by a root local rather than shoot generated factor(s). Importantly, pdx1 suppresses yucca mutant, an auxin overproducer, in root hair proliferation although it fails to suppress the hypocotyl elongation phenotype.20 Our work thus demonstrated that pdx1 has impaired root local auxin biosynthesis from tryptophan. To test whether the synthesis of tryptophan is also affected in pdx1 mutant, we planted pdx1 together with wild-type seeds on Murashige and Skoog (MS) medium supplemented with 5-mehtyl-anthranilate (5-MA), an analogue of the Trp biosynthetic intermediate anthranilate.21 Although pdx1 seedlings grew poorly under the control conditions, the growth of wild-type seedlings was more inhibited than that of the pdx1 seedlings on 10 µM 5-MA media (Fig. 1A–D). Compared with the elongated primary root on MS, wild-type seedlings showed very limited root growth on 5-MA (Fig. 1E). The relatively increased tolerance to 5-MA of pdx1 thus indicates that the pdx1 mutant may be defective in Trp biosynthesis, although amino acid analysis of the bulked seedlings did not find clear changes in Trp levels in the mutants (our unpublished data).Open in a separate windowFigure 1The pdx1 mutant seedlings are relatively less sensitive to toxic 5-methyl anthranilate (5-MA). (A and C) Five-day-old seedlings of the wild type (Col-0) (A) or pdx1 (C) on MS medium. (B and D) Five-day-old seedlings of the wild type (B) or pdx1 (D) on MS medium supplemented with 10 µM 5-MA. (E) Eight-day-old seedlings of the wild type or pdx1 on MS medium without or with 10 µM 5-MA supplement. Sterilized seeds were planted directly on the indicated medium and after two days of cold treatment, the plates were incubated under continuous light at 22–24°C before taking pictures.We reported that PDX1 is required for tolerance to oxidative stresses in Arabidopsis.19 Interestingly, redox homeostasis appears to play a critical role in Arabidopsis root development. The glutathione-deficient mutant root meristemless1 (rml1) and the vitamin C-deficient mutant vitamin C1 (vtc1) both have similar stunted roots.22,23 Nonetheless, pdx1 is not rescued by either glutathione or vitamin C19 suggesting that the pdx1 short-root phenotype may not be resulted from a general reduction of antioxidative capacity. Interestingly, ascorbate oxidase is found to be highly expressed in the maize root quiescent center.24 This enzyme can oxidatively decarboxylate auxin in vitro, suggesting that the quiescent center may be a site for metabolizing auxin to control its homeostasis.25 It is therefore likely that the reduced auxin level in pdx1 root tips could be partially caused by increased auxin catabolism resulted from reduced vitamin B6 level. We thus conducted experiments to test this possibility. A quiescent center-specific promoter WOX5 driven bacterial auxin biosynthetic gene iaaH26 was introduced into pdx1 mutant. The transgenic seeds were planted on media supplemented with different concentrations of indoleacetamide (IAM), the substrate of iaaH protein. Although promotion of lateral root growth was observed at higher IAM concentrations, which indicates increased tryptophan-independent auxin production from the transgene, no change in root elongation was observed between pdx1 with or without the WOX5::iaaH transgene at any concentration of IAM tested (data not shown), suggesting that the pdx1 short-root phenotype may not be due to increased auxin catabolism.Taken together, in addition to auxin transport; temporally, spatially or developmentally coordinated local auxin biosynthesis defines the plant growth and its response to environmental changes.8,14,15  相似文献   

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Thioredoxin (NTR/TRX) and glutathione (GSH/GRX) are the two major systems that play a key role in the maintenance of cellular redox homeostasis. They are essential for plant development, cell division or the response to environmental stresses. In a recent article,1 we studied the interplay between the NADP-linked thioredoxin and glutathione systems in auxin signaling genetically, by associating TRX reductase (ntra ntrb) and glutathione biosynthesis (cad2) mutations. We show that these two thiol reduction pathways interfere with developmental processes. This occurs through modulation of auxin activity as shown by genetic analyses of loss of function mutations in a triple ntra ntrb cad2 mutant. The triple mutant develops almost normally at the rosette stage but fails to generate lateral organs from the inflorescence meristem, producing almost naked stems that are reminiscent of mutants affected in PAT (polar auxin transport) or biosynthesis. The triple mutant exhibits other defects in processes regulated by auxin, including a loss of apical dominance, vasculature defects and reduced secondary root production. Furthermore, it has lower auxin (IAA) levels and decreased capacity for PAT, suggesting that the NTR and glutathione pathways influence inflorescence meristem development through regulation of auxin transport and metabolism.Key words: arabidopsis, NTS pathway, NGS pathway, thioredoxin (TRX), glutaredoxine (GRX), polar auxin transport (PAT), auxin biosynthesis, pin-like phenotype, apical dominance, meristematic activityExposure of living organisms to environmental stresses triggers various defense and developmental responses. Redox signaling is involved in many aspects of these responses.26 The key players in these responses are the NADPH-dependent glutathione/glutaredoxin system (NGS) and the NADPH-dependent thioredoxin system (NTS). TRX and GRX play key roles in the maintenance of cellular redox homeostasis.710 Genetic approaches aiming to identify functions of TRX and GRX in knock-out plants have largely been limited by the absence of phenotypes of single mutants, presumably due to functional redundancies among members of the multigene families of TRX and GRX.11 Interplay between NTS and NGS pathways have been studied in different organisms1217 and association of mutants involved in these two pathways have recently revealed new functions in several aspects of plant development.46  相似文献   

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A role for SR proteins in plant stress responses   总被引:1,自引:0,他引:1  
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Hormones typically serve as long distance signaling molecules. To reach their site of action, hormones need to be transported from the sites of synthesis. Many plant hormones are mobile, thus requiring specific transport systems for the export from their source cells as well as subsequent import into target cells. Hormone transport in general is still poorly understood. Auxin is probably the most intensively studied plant hormone concerning transport in the moment. To advance our understanding of hormone transport we need two principal data sets: information on the properties of the transport systems including substrate specificity and kinetics, and we need to identify candidate genes for the respective transporters. Physiological transport data can provide an important basis for identifying and characterizing candidate transporters and to define their in vivo role. A recent publication in Plant Physiology highlights how kinetic and specificity studies may help to identify cytokinin transporters.1Key words: kinetin, zeatin, adenine, phytohormone, transportBy definition, hormones are compounds that interact at low concentrations with cellular receptors to modulate signal transduction pathways. A comparison of the chemical structures of animal and plant hormones suggests potential common origins. Peptide hormones are found in both kingdoms and share common processing mechanisms (e.g., TRH, vasopressin and kinins in animals; systemins, phytosulfokines, self incompatibility peptides in plants).2,3 Steroid hormones derived from cholesterol such as testosterone, cortisol and calcitriol regulate development in mammals; the steroid hormone brassinolide is essential for plant development.4 Glutamate can serve as metabolite and signal in both plants and animals.5,6 Finally, lipid and phospholipid-derived signaling compounds such as linoleic acid and arachidonic acid also function in both plants and animals; with phospholipid-derived prostaglandins and eicosanoids bearing similarities to the plant defense compound jasmonic acid.7Other signaling compounds present in animals have yet to be shown to function in plants, e.g., glycoprotein hormones such as luteinizing hormone, follicle-stimulating hormone or thyroid-stimulating hormone have been not been described to exist in plants.8 Compounds structurally similar to animal amine-derived hormones derived from tyrosine and tryptophan (such as catecholamines and thyroxine) are also present in plants, but appear to function primarily in herbivore defense.9The best characterized, and arguably most important plant hormones, bear little similarity to animal hormones and are mechanistically distinct. These include auxins, cytokinins, gibberellins, abscisic acid, ethylene and an apparent carotenoid-derivative, the MAX-dependent regulator of auxin signaling.10,11 Arguably, the stress response compound salicylic acid, which functions in stress, wounding and defense responses could also be considered a plant hormone.12Hormonal signaling mechanisms can be categorized as autocrine (acting at the site of biosynthesis), paracrine (acting in adjacent or proximal cells), and endocrine (acting in cells distal to the site of production). In both, plants and animals, paracrine and endocrine hormone action is mediated and influenced by multiple long distance delivery systems. Hormones move primarily through the circulatory system in animals, but, in plants, are mobilized by transpiration and source-sink flows, which can be directed by chemisomotically-driven cellular uptake and efflux. However, the mechanisms driving uptake and efflux at the cellular level, as well as the proteins that mediate this movement, are surprisingly similar in plants and animals, despite the dissimilarities of plant and animal cell structure (central vacuoles, cell walls and H+ versus K+/Na2+ in/out gradients).Surprisingly little is known about plant hormone transport. Most hormones have autocrine activity, but in order to act at a distance or to even act on adjacent cells they must be transported across membranes. The existence of cellular export and import mechanisms are suggested by the presence of multiple hormones in the phloem sap13,14 and the well documented polar long distance movement of auxin.15 Brassinosteroid receptors have been demonstrated as integral plasma membrane proteins which receive the hormone signal from outside the cell.16 This suggests a need for the hormone to first move into the apoplasm after biosynthesis. However, until recently, only the cellular auxin transport mechanisms mediated by the AUX/LAX, PIN and AtABCB/PGP proteins has been well characterized (reviewed in ref. 17).The study of these transporters has benefited from the use of plant, yeast and animal expression systems to characterize the proteins involved. Analyses of auxin transport proteins have capitalized on earlier suppression cloning and radiotracer uptake studies used successfully to characterize ion and metabolite transporters in yeast.1821 In cases where yeast systems have proven intractable for analysis of auxin transport proteins, heterologous systems based on mammalian cell systems have proven to be highly effective for radiotracer uptake studies.1823 Xenopus oocyte expression has been successfully utilized to characterize the AUX/LAX family of auxin influx symporters.24,25 Plant cell culture systems have also been used to characterize transport proteins. This can however be problematic when endogenous substrates are metabolized by the cells, as is the case with IAA in tobacco BY-2 and Arabidopsis cell cultures.19 It is also difficult to assess the function of plant proteins in undifferentiated cell cultures, which may differ from the native function in phloem or xylem parenchyma cells.A recent article describes the use of a heterologous expression system based on the fission yeast S. pombe to express and characterize the PIN1 auxin efflux protein after knock-out of the endogenous yeast PIN-like gene AEL1.21 Previously, PIN1 had only been functionally expressed in plant cell systems and was nonfunctional when expressed in baker''s yeast or mammalian cells.19,22 This report suggests that PIN1, interacts synergistically with the AtABCB19/PGP19 auxin efflux transporter, but appears to also mediate auxin efflux on its own, consistent with the distant phylogenetic similarity of the auxin efflux transporter protein family to major facilitator proteins.Subsequent work in the Murphy lab has shown that S. pombe can be used for comparisons of all known auxin transporters in a single system in which all ABC transporters and a solitary AUX1-like gene had been knocked out (Yang and Murphy, unpublished). This system also allows for the more detailed analyses of substrate specificity, transport kinetics and coupling mechanisms (primary and secondary active transport, uniport, cotransport antiport) necessary for functional assignment of auxin transport proteins. This system may also provide an attractive alternative to baker''s yeast when functional expression of a plant protein in Saccharomyces cerevisiae proves unsuccessful.Similar efforts are required for characterizing the transport of all other plant hormones including cytokinin. Arabidopsis transporters mediating both trans-zeatin and adenine uptake had been identified using yeast as an expression system.26 Recently, the Schulz and Frommer labs provided a reference data set for trans-zeatin uptake by characterizing radiolabeled trans-zeatin uptake in Arabidopsis cell cultures.1 The data show that the uptake kinetics of trans-zeatin are multiphasic, indicating the presence of both low- and high-affinity transport systems. The protonophore CCCP is an effective inhibitor of cytokinin uptake, consistent with H+-mediated uptake. Other physiologically active cytokinins such as isopentenyladenine and benzylaminopurine are effective competitors of trans-zeatin uptake, whereas allantoin had no inhibitory effect. Adenine competes for zeatin uptake indicating that degradation products of cytokinin oxidases can be transported by the same systems. Comparison of adenine and trans-zeatin uptake in Arabidopsis seedlings reveals similar uptake kinetics. Kinetic properties as well as substrate specificity determined in cell cultures are compatible with the hypothesis that members of the plant-specific PUP transporter family may play a role in adenine transport to scavenge extracellular adenine. In addition, the findings are also compatible with the hypothesis that this class of transporters may be involved at least in low affinity (µM range) cytokinin uptake. PUPs are encoded by a large gene family of 21 members, so it is conceivable that other members of the family may be involved in high affinity transport. Systematic analyses of single knock outs in Arabidopsis and combinations thereof my help to shed more light on the role of PUPs in cytokinin transport.  相似文献   

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The molecularly well-characterized auxin signal transduction pathway involves two evolutionarily conserved families interacting through their C-terminal domains III and IV: the Auxin Response Factors (ARFs) and their repressors the Aux/IAAs, to control auxin-responsive genes, among them genes involved in auxin transport.1,2 We have developed a new genetic tool to study ARF function. Using MONOPTEROS (MP)/ARF5, we have generated a truncated version of MP (MPΔ),3 which has lost the target domains for repression by Aux/IAA proteins. Besides exploring genetic interactions between MP and Aux/IAAs, we used this construct to trace MP’s role in vascular patterning, a previously characterized auxin dependent process.4,5 Here we summarize examples of naturally occurring truncated ARFs and summarize potential applications of truncated ARFs as analytical tools.  相似文献   

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The pathogenicity of Clostridium difficile (C. difficile) is mediated by the release of two toxins, A and B. Both toxins contain large clusters of repeats known as cell wall binding (CWB) domains responsible for binding epithelial cell surfaces. Several murine monoclonal antibodies were generated against the CWB domain of toxin A and screened for their ability to neutralize the toxin individually and in combination. Three antibodies capable of neutralizing toxin A all recognized multiple sites on toxin A, suggesting that the extent of surface coverage may contribute to neutralization. Combination of two noncompeting antibodies, denoted 3358 and 3359, enhanced toxin A neutralization over saturating levels of single antibodies. Antibody 3358 increased the level of detectable CWB domain on the surface of cells, while 3359 inhibited CWB domain cell surface association. These results suggest that antibody combinations that cover a broader epitope space on the CWB repeat domains of toxin A (and potentially toxin B) and utilize multiple mechanisms to reduce toxin internalization may provide enhanced protection against C. difficile-associated diarrhea.Key words: Clostridium difficile, toxin neutralization, therapeutic antibody, cell wall binding domains, repeat proteins, CROPs, mAb combinationThe most common cause of nosocomial antibiotic-associated diarrhea is the gram-positive, spore-forming anaerobic bacillus Clostridium difficile (C. difficile). Infection can be asymptomatic or lead to acute diarrhea, colitis, and in severe instances, pseudomembranous colitis and toxic megacolon.1,2The pathological effects of C. difficile have long been linked to two secreted toxins, A and B.3,4 Some strains, particularly the virulent and antibiotic-resistant strain 027 with toxinotype III, also produce a binary toxin whose significance in the pathogenicity and severity of disease is still unclear.5 Early studies including in vitro cell-killing assays and ex vivo models indicated that toxin A is more toxigenic than toxin B; however, recent gene manipulation studies and the emergence of virulent C. difficile strains that do not express significant levels of toxin A (termed “A B+”) suggest a critical role for toxin B in pathogenicity.6,7Toxins A and B are large multidomain proteins with high homology to one another. The N-terminal region of both toxins enzymatically glucosylates small GTP binding proteins including Rho, Rac and CDC42,8,9 leading to altered actin expression and the disruption of cytoskeletal integrity.9,10 The C-terminal region of both toxins is composed of 20 to 30 residue repeats known as the clostridial repetitive oligopeptides (CROPs) or cell wall binding (CWB) domains due to their homology to the repeats of Streptococcus pneumoniae LytA,1114 and is responsible for cell surface recognition and endocytosis.12,1517C. difficile-associated diarrhea is often, but not always, induced by antibiotic clearance of the normal intestinal flora followed by mucosal C. difficile colonization resulting from preexisting antibiotic resistant C. difficile or concomitant exposure to C. difficile spores, particularly in hospitals. Treatments for C. difficile include administration of metronidazole or vancomycin.2,18 These agents are effective; however, approximately 20% of patients relapse. Resistance of C. difficile to these antibiotics is also an emerging issue19,20 and various non-antibiotic treatments are under investigation.2025Because hospital patients who contract C. difficile and remain asymptomatic have generally mounted strong antibody responses to the toxins,26,27 active or passive immunization approaches are considered hopeful avenues of treatment for the disease. Toxins A and B have been the primary targets for immunization approaches.20,2833 Polyclonal antibodies against toxins A and B, particularly those that recognize the CWB domains, have been shown to effectively neutralize the toxins and inhibit morbidity in rodent infection models.31 Monoclonal antibodies (mAbs) against the CWB domains of the toxins have also demonstrated neutralizing capabilities; however, their activity in cell-based assays is significantly weaker than that observed for polyclonal antibody mixtures.3336We investigated the possibility of creating a cocktail of two or more neutralizing mAbs that target the CWB domain of toxin A with the goal of synthetically re-creating the superior neutralization properties of polyclonal antibody mixtures. Using the entire CWB domain of toxin A, antibodies were raised in rodents and screened for their ability to neutralize toxin A in a cell-based assay. Two mAbs, 3358 and 3359, that (1) both independently demonstrated marginal neutralization behavior and (2) did not cross-block one another from binding toxin A were identified. We report here that 3358 and 3359 use differing mechanisms to modify CWB-domain association with CHO cell surfaces and combine favorably to reduce toxin A-mediated cell lysis.  相似文献   

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Polar auxin transport (PAT), which is controlled precisely by both auxin efflux and influx facilitators and mediated by the cell trafficking system, modulates organogenesis, development and root gravitropism. ADP-ribosylation factor (ARF)-GTPase protein is catalyzed to switch to the GTP-bound type by a guanine nucleotide exchange factor (GEF) and promoted for hybridization to the GDP-bound type by a GTPase-activating protein (GAP). Previous studies showed that auxin efflux facilitators such as PIN1 are regulated by GNOM, an ARF-GEF, in Arabidopsis. In the November issue of The Plant Journal, we reported that the auxin influx facilitator AUX1 was regulated by ARF-GAP via the vesicle trafficking system.1 In this addendum, we report that overexpression of OsAGAP leads to enhanced root gravitropism and propose a new model of PAT regulation: a loop mechanism between ARF-GAP and GEF mediated by vesicle trafficking to regulate PAT at influx and efflux facilitators, thus controlling root development in plants.Key Words: ADP-ribosylation factor (ARF), ARF-GAP, ARF-GEF, auxin, GNOM, polar transport of auxinPolar auxin transport (PAT) is a unique process in plants. It results in alteration of auxin level, which controls organogenesis and development and a series of physiological processes, such as vascular differentiation, apical dominance, and tropic growth.2 Genetic and physiological studies identified that PAT depends on efflux facilitators such as PIN family proteins and influx facilitators such as AUX1 in Arabidopsis.Eight PIN family proteins, AtPIN1 to AtPIN8, exist in Arabidopsis. AtPIN1 is located at the basal side of the plasma membrane in vascular tissues but is weak in cortical tissues, which supports the hypothesis of chemical pervasion.3 AtPIN2 is localized at the apical side of epidermal cells and basally in cortical cells.1,4 GNOM, an ARF GEF, modulates the localization of PIN1 and vesicle trafficking and affects root development.5,6 The PIN auxin-efflux facilitator network controls root growth and patterning in Arabidopsis.4 As well, asymmetric localization of AUX1 occurs in the root cells of Arabidopsis plants,7 and overexpression of OsAGAP interferes with localization of AUX1.1 Our data support that ARF-GAP mediates auxin influx and auxin-dependent root growth and patterning, which involves vesicle trafficking.1 Here we show that OsAGAP overexpression leads to enhanced gravitropic response in transgenic rice plants. We propose a model whereby ARF GTPase is a molecular switch to control PAT and root growth and development.Overexpression of OsAGAP led to reduced growth in primary or adventitious roots of rice as compared with wild-type rice.1 Gravitropism assay revealed transgenic rice overxpressing OsAGAP with a faster response to gravity than the wild type during 24-h treatment. However, 1-naphthyl acetic acid (NAA) treatment promoted the gravitropic response of the wild type, with no difference in response between the OsAGAP transgenic plants and the wild type plants (Fig. 1). The phenotype of enhanced gravitropic response in the transgenic plants was similar to that in the mutants atmdr1-100 and atmdr1-100/atpgp1-100 related to Arabidopsis ABC (ATP-binding cassette) transporter and defective in PAT.8 The physiological data, as well as data on localization of auxin transport facilitators, support ARF-GAP modulating PAT via regulating the location of the auxin influx facilitator AUX1.1 So the alteration in gravitropic response in the OsAGAP transgenic plants was explained by a defect in PAT.Open in a separate windowFigure 1Gravitropism of OsAGAP overexpressing transgenic rice roots and response to 1-naphthyl acetic acid (NAA). (A) Gravitropism phenotype of wild type (WT) and OsAGAP overexpressing roots at 6 hr gravi-stimulation (top panel) and 0 hr as a treatment control (bottom panel). (B) Time course of gravitropic response in transgenic roots. (C and D) results correspond to those in (A and B), except for treatment with NAA (5 × 10−7 M).The polarity of auxin transport is controlled by the asymmetric distribution of auxin transport proteins, efflux facilitators and influx carriers. ARF GTPase is a key member in vesicle trafficking system and modulates cell polarity and PAT in plants. Thus, ARF-GDP or GTP bound with GEF or GAP determines the ARF function on auxin efflux facilitators (such as PIN1) or influx ones (such as AUX1).ARF1, targeting ROP2 and PIN2, affects epidermal cell polarity.9 GNOM is involved in the regulation of PIN1 asymmetric localization in cells and its related function in organogenesis and development.6 Although VAN3, an ARF-GAP in Arabidopsis, is located in a subpopulation of the trans-Golgi transport network (TGN), which is involved in leaf vascular network formation, it does not affect PAT.10 OsAGAP possesses an ARF GTPase-activating function in rice.11 Specifically, our evidence supports that ARF-GAP bound with ARF-GTP modulates PAT and gravitropism via AUX1, mediated by vesicle trafficking, including the Golgi stack.1Therefore, we propose a loop mechanism between ARF-GAP and GEF mediated by the vascular trafficking system in regulating PAT at influx and efflux facilitators, which controls root development and gravitropism in plants (Fig. 2). Here we emphasize that ARF-GEF catalyzes a conversion of ARF-bound GDP to GTP, which is necessary for the efficient delivery of the vesicle to the target membrane.12 An opposite process of ARF-bound GDP to GTP is promoted by ARF-GTPase-activating protein via binding. A loop status of ARF-GTP and ARF-GDP bound with their appurtenances controls different auxin facilitators and regulates root development and gravitropism.Open in a separate windowFigure 2Model for ARF GTPase as a molecular switch for the polar auxin transport mediated by the vesicle traffic system.  相似文献   

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