RESUMO
Pea phytoalexins (-)-maackiain and (+)-pisatin have opposite C6a/C11a configurations, but biosynthetically how this occurs is unknown. Pea dirigent-protein (DP) PsPTS2 generates 7,2'-dihydroxy-4',5'-methylenedioxyisoflav-3-ene (DMDIF), and stereoselectivity toward four possible 7,2'-dihydroxy-4',5'-methylenedioxyisoflavan-4-ol (DMDI) stereoisomers was investigated. Stereoisomer configurations were determined using NMR spectroscopy, electronic circular dichroism, and molecular orbital analyses. PsPTS2 efficiently converted cis-(3R,4R)-DMDI into DMDIF 20-fold faster than the trans-(3R,4S)-isomer. The 4R-configured substrate's near ß-axial OH orientation significantly enhanced its leaving group abilities in generating A-ring mono-quinone methide (QM), whereas 4S-isomer's α-equatorial-OH was a poorer leaving group. Docking simulations indicated that the 4R-configured ß-axial OH was closest to Asp51, whereas 4S-isomer's α-equatorial OH was further away. Neither cis-(3S,4S)- nor trans-(3S,4R)-DMDIs were substrates, even with the former having C3/C4 stereochemistry as in (+)-pisatin. PsPTS2 used cis-(3R,4R)-7,2'-dihydroxy-4'-methoxyisoflavan-4-ol [cis-(3R,4R)-DMI] and C3/C4 stereoisomers to give 2',7-dihydroxy-4'-methoxyisoflav-3-ene (DMIF). DP homologs may exist in licorice (Glycyrrhiza pallidiflora) and tree legume Bolusanthus speciosus, as DMIF occurs in both species. PsPTS1 utilized cis-(3R,4R)-DMDI to give (-)-maackiain 2200-fold more efficiently than with cis-(3R,4R)-DMI to give (-)-medicarpin. PsPTS1 also slowly converted trans-(3S,4R)-DMDI into (+)-maackiain, reflecting the better 4R configured OH leaving group. PsPTS2 and PsPTS1 provisionally provide the means to enable differing C6a and C11a configurations in (+)-pisatin and (-)-maackiain, via identical DP-engendered mono-QM bound intermediate generation, which PsPTS2 either re-aromatizes to give DMDIF or PsPTS1 intramolecularly cyclizes to afford (-)-maackiain. Substrate docking simulations using PsPTS2 and PsPTS1 indicate cis-(3R,4R)-DMDI binds in the anti-configuration in PsPTS2 to afford DMDIF, and the syn-configuration in PsPTS1 to give maackiain.
Assuntos
Pisum sativum , Proteínas de Plantas , Pterocarpanos , Pisum sativum/química , Pisum sativum/metabolismo , Pterocarpanos/química , Pterocarpanos/metabolismo , Estereoisomerismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Modelos Moleculares , Conformação MolecularRESUMO
PREMISE OF THE STUDY: Root border cells are programmed to separate from the root cap as it penetrates the soil environment, where the cells actively secrete >100 extracellular proteins into the surrounding mucilage. The detached cells function in defense of the root tip by an extracellular trapping process that also requires DNA, as in mammalian white blood cells. Trapping in animals and plants is reversed by treatment with DNase, which results in increased infection. The goal of this study was to evaluate the role of DNA in the structural integrity of extracellular structures released as border cells disperse from the root tip upon contact with water. METHODS: DNA stains including crystal violet, toluidine blue, Hoechst 33342, DAPI, and SYTOX green were added to root tips to visualize the extracellular mucilage as it absorbed water and border cell populations dispersed. DNase I was used to assess structural changes occurring when extracellular DNA was degraded. KEY RESULTS: Complex masses associated with living border cells were immediately evident in response to each stain, including those that are specific for DNA. Treating with DNase I dramatically altered the appearance of the extracellular structures and their association with border cells. No extracellular DNA was found in association with border cells killed by freezing or high-speed centrifugation. This observation is consistent with the hypothesis that, as with border cell extracellular proteins, DNA is secreted by living cells. CONCLUSION: DNA is an integral component of border cell extracellular traps.
Assuntos
DNA de Plantas/química , Meristema/citologia , Pisum sativum/citologia , Raízes de Plantas/citologia , Zea mays/citologia , Meristema/crescimento & desenvolvimento , Pisum sativum/crescimento & desenvolvimento , Raízes de Plantas/crescimento & desenvolvimento , Zea mays/crescimento & desenvolvimentoRESUMO
PREMISE OF THE STUDY: Border cells, which separate from the root cap, can comprise >90% of carbon-based exudates released into the rhizosphere, but may not provide a general source of nutrients for soil microorganisms. Instead, this population of specialized cells appears to function in defense of the root tip by an extracellular trapping process similar to that of mammalian white blood cells. Border cell production is tightly regulated, and direct tests of their impact on crop production have been hindered by lack of intraspecies variation. ⢠METHODS: Border cell number, viability, and clumping were compared among 22 cotton cultivars. Slime layer "extracellular trap" production by border cells in response to copper chloride, an elicitor of plant defenses, was compared in two cultivars with divergent border cell production. Trapping of bacteria by border cells in these lines also was measured. ⢠KEY RESULTS: Emerging roots of some cultivars produced more than 20000 border cells per root, a 100% increase over previously reported values for this species. No differences in border cell morphology, viability, or clumping were found. Copper chloride-induced extracellular trap formation by border cells from a cultivar that produced 27921 ± 2111 cells per root was similar to that of cells from a cultivar with 10002 ± 614 cells, but bacterial trapping was reduced. ⢠CONCLUSIONS: Intraspecific variation in border cell production provides a tool to measure their impact on plant development in the laboratory, greenhouse, and field. Further research is needed to determine the basis for this variation, and its impact on rhizosphere community structure.
Assuntos
Bacillus subtilis/fisiologia , Gossypium/fisiologia , Interações Hospedeiro-Patógeno , Pectobacterium carotovorum/fisiologia , Raízes de Plantas/fisiologia , Gossypium/citologia , Gossypium/crescimento & desenvolvimento , Gossypium/microbiologia , Meristema/crescimento & desenvolvimento , Meristema/microbiologia , Meristema/fisiologia , Fenótipo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Rizosfera , Especificidade da EspécieRESUMO
Commercial application of compost to prevent plant disease is hindered by variable performance. Here, we describe the use of a growth pouch assay to measure impact of a compost water extract (CWE) on root infection under controlled conditions. Most pea roots (≥95%) inoculated with Fusarium solani or Phoma pinodella spores rapidly develop a single local lesion in the region of elongation. In the presence of CWE, infection of pea roots grown in pouches was reduced by 93 to 100%. CWE used as a drench on pea seedlings grown in sand also resulted in 100% protection but, in a heavy clay soil, infection was reduced by <50%. CWE filtered to remove microorganisms did not inhibit frequency of F. solani infection, and resulted in increased local lesion development on individual roots. CWE inhibited mycelial growth of both pea- and cucumber-infecting isolates of F. solani in culture but exerted <40% protection against cucumber root infection. CWE treatment of pea but not cucumber was associated with retention of a sheath of border cells interspersed with bacteria covering the region of elongation. Growth pouch assays may provide a system to monitor effects of specific compost mixtures on root-rhizosphere interactions, and to identify variables influencing disease control.
Assuntos
Cucumis sativus/imunologia , Fusarium/patogenicidade , Pisum sativum/imunologia , Doenças das Plantas/imunologia , Raízes de Plantas/imunologia , Ascomicetos/crescimento & desenvolvimento , Ascomicetos/patogenicidade , Produtos Agrícolas , Cucumis sativus/microbiologia , Cucumis sativus/fisiologia , Suscetibilidade a Doenças , Fusarium/crescimento & desenvolvimento , Pisum sativum/microbiologia , Pisum sativum/fisiologia , Doenças das Plantas/microbiologia , Raízes de Plantas/microbiologia , Raízes de Plantas/fisiologia , Plântula/imunologia , Plântula/microbiologia , Plântula/fisiologia , Solo/química , ÁguaRESUMO
PREMISE OF THE STUDY: Lead (Pb) is a contaminant whose removal from soil remains a challenge. In a previous study, border cells released from root tips were found to trap Pb, alter its chemistry, and prevent root uptake. Rhodizonic acid (RA) is a forensic tool used to reveal gunshot residue, and also to detect Pb within plant tissues. Here we report preliminary observations to assess the potential application of RA in exploring the dynamics of Pb accumulation at the root tip surface. METHODS AND RESULTS: Corn root tips were immersed in Pb solution, stained with RA, and observed microscopically. Pb trapping by border cells was evident within minutes. The role of extracellular DNA was revealed when addition of nucleases resulted in dispersal of RA-stained Pb particles. CONCLUSIONS: RA is an efficient tool to monitor Pb-root interactions. Trapping by border cells may control Pb levels and chemistry at the root tip surface. Understanding how plants influence Pb distribution in soil may facilitate its remediation.
RESUMO
Histone-linked extracellular DNA (exDNA) is a component of neutrophil extracellular traps (NETs). NETs have been shown to play a role in immune response to bacteria, fungi, viruses, and protozoan parasites. Mutation of genes encoding group A Streptococcus extracellular DNases (exDNases) results in reduced virulence in animals, a finding that implies that exDNases are deployed as counter defense against host DNA-containing NETs. Is the exDNA/exDNase mechanism also relevant to plants and their pathogens? It has been demonstrated previously that exDNA is a component of a matrix secreted from plant root caps and that plants also carry out an extracellular trapping process. Treatment with DNase I destroys root tip resistance to infection by fungi, the most abundant plant pathogens. We show that the absence of a single gene encoding a candidate exDNase results in significantly reduced virulence of a fungal plant pathogen to its host on leaves, the known infection site, and on roots. Mg2+-dependent exDNase activity was demonstrated in fungal culture filtrates and induced when host leaf material was present. It is speculated that the enzyme functions to degrade plant-secreted DNA, a component of a complex matrix akin to neutrophil extracellular traps of animals.IMPORTANCE We document that the absence of a single gene encoding a DNase in a fungal plant pathogen results in significantly reduced virulence to a plant host. We compared a wild-type strain of the maize pathogen Cochliobolus heterostrophus and an isogenic mutant lacking a candidate secreted DNase-encoding gene and demonstrated that the mutant is reduced in virulence on leaves and on roots. There are no previous reports of deletion of such a gene from either an animal or plant fungal pathogen accompanied by comparative assays of mutants and wild type for alterations in virulence. We observed DNase activity, in fungal culture filtrates, that is Mg2+ dependent and induced when plant host leaf material is present. Our findings demonstrate not only that fungi use extracellular DNases (exDNases) for virulence, but also that the relevant molecules are deployed in above-ground leaves as well as below-ground plant tissues. Overall, these data provide support for a common defense/counter defense virulence mechanism used by animals, plants, and their fungal and bacterial pathogens and suggest that components of the mechanism might be novel targets for the control of plant disease.
Assuntos
Ascomicetos/enzimologia , Ascomicetos/crescimento & desenvolvimento , DNA de Plantas/metabolismo , Desoxirribonucleases/metabolismo , Interações Hospedeiro-Patógeno , Doenças das Plantas/microbiologia , Fatores de Virulência/metabolismo , Animais , Hidrólise , Folhas de Planta/microbiologia , Raízes de Plantas/microbiologia , Zea maysRESUMO
PURPOSE: To evaluate the hypothesis that spinal fusion surgery is an effective method to address spinal deformity-associated clinical problems, including magnitude of curvature (Cobb angle), pulmonary dysfunction, and pain. METHOD: A systematic review was carried out using Science Citation Index (SCI) Expanded (1900 - present), Social Sciences Citation Index (1956 - present), Arts and Humanities Citation Index (1965 - present), Medline (1950 - present) and PubMed Central databases (1887 - present) to access information regarding efficacy of spine surgery in preventing or improving the health and function of patients diagnosed with scoliosis in adolescence. RESULTS: Since 1950, more than 12,600 articles on scoliosis have been published, and nearly 50% (5721) focus on methods, rationale, outcome, and complications of surgical intervention. Among these, 82 articles have documented outcome for groups of > or =10 patients, treated for adolescent idiopathic scoliosis, and followed for at least 2 years after treatment. These data provide an overview of the impact of spine surgery on scoliosis for 5780 patients as surgery methods and approaches have evolved. CONCLUSIONS: For most patients, a reduced magnitude of spinal curvature can be achieved through one or more spinal fusion surgeries. There is no evidence to support the premise that this result is correlated with improved pulmonary function or reduced pain.
Assuntos
Escoliose/cirurgia , Fusão Vertebral , Adolescente , Adulto , Seguimentos , Humanos , Medição da Dor , Satisfação do Paciente , Resultado do TratamentoRESUMO
Understanding the cause of a disease or disorder is key to developing effective and humane strategies for early intervention and treatment. School screening programs have made it possible to demonstrate the high prevalence of childhood scoliosis, worldwide, and to reliably identify spinal curvatures early in the disease process before progression to a fixed structural deformity. Unfortunately, effective early interventions have not been established. Developing strategies to prevent scoliosis has been compromised, in general, by lack of understanding of its causes on a case by case basis. Information about genetic loci associated with disorders including scoliosis is emerging rapidly, since completion of the human genome sequence in 2003. These data can be used to identify children at high risk for developing spinal deformities and to design strategies for prevention.
Assuntos
Projeto Genoma Humano , Escoliose/genética , HumanosRESUMO
This guideline has been discussed by the SOSORT guideline committee prior to the SOSORT consensus meeting in Milan, January 2005 and published in its first version on the SOSORT homepage: http://www.sosort.org/meetings.php. After the meeting it again has been discussed by the members of the SOSORT guideline committee to establish the final 2005 version submitted to Scoliosis, the official Journal of the society, in December 2005. This chapter is a republication from the original paper published in "Scoliosis" BioMed journal and it is included in this book due to its high importance.
Assuntos
Protocolos Clínicos , Escoliose/terapia , Adolescente , Guias como Assunto , Humanos , Escoliose/classificação , Escoliose/epidemiologia , Escoliose/etiologiaRESUMO
DNase I is a secreted enzyme whose function has been presumed to control "waste management" in the human system, by degrading DNA that leaks from dead and dying cells. Emerging studies have instead yielded evidence that DNase I plays a central role in newly defined dynamics of immune and autoimmune diseases, as well as cancer and vascular disorders, including thrombosis. Cancer cells have been reported to be associated with distinctive extracellular structures that facilitate aggregation and implantation. The fact that DNA is a component of such structures and that it plays a role in cancer development is illustrated by direct evidence: DNase I added to tumor cells eliminates the structures and inhibits tumorigenicity of some cancer cell lines. DNase I injected into experimental animals, moreover, results in significant inhibition of metastasis. Despite independent observations of such phenomena in diverse cancers for over 50 years, the potential for using DNase I as a clinical tool to prevent or treat cancer remains unexplored. The discovery of neutrophil extracellular traps has yielded a conceptual framework for interpreting how extracellular DNA may function in cancer development and why it may prove to be an important clinical target in stopping cancer outside the cell.
Assuntos
DNA/metabolismo , Neoplasias/etiologia , Neoplasias/metabolismo , Estudos de Casos e Controles , Desoxirribonuclease I/metabolismo , Desoxirribonucleases/metabolismo , Espaço Extracelular/genética , Espaço Extracelular/metabolismo , Armadilhas Extracelulares/genética , Armadilhas Extracelulares/metabolismo , HumanosRESUMO
When roots of pea seedlings were inoculated uniformly with spores of Nectria haematocca or other pea pathogenic fungi, more than 90% developed lesions in the region of elongation within 3 days. More mature regions of most roots as well as the tip showed no visible signs of infection. Yet, microscopic observation revealed that 'mantles,' comprised of fungal hyphae intermeshed with populations of border cells, covered the tips of most roots. After physical detachment of the mantle, the underlying tip of most roots was found to be free of infection. Mantle-covered root tips did not respond to invasion of their border cells by activation of known defense genes unless there was invasion of the tip itself, as revealed by the presence of a lesion. Concomitant with the activation of defense genes was the induction of a cell-wall degrading enzyme whose expression is a marker for renewed production of border cells. Mantle formation did not occur in response to nonpathogens. The data are consistent with the hypothesis that border cells serve as a host-specific 'decoy' that protects root meristems by inhibiting fungal infection of the root tip.
Assuntos
Ascomicetos/crescimento & desenvolvimento , Meristema/genética , Doenças das Plantas/genética , Raízes de Plantas/genética , Regulação da Expressão Gênica de Plantas , Imunidade Inata/genética , Meristema/citologia , Meristema/microbiologia , Pisum sativum/genética , Pisum sativum/microbiologia , Doenças das Plantas/microbiologia , Raízes de Plantas/citologia , Raízes de Plantas/microbiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
This report describes improved signs and symptoms of previously untreated symptomatic spinal deformity in an adult female diagnosed with moderately severe thoracic scoliosis at the age of .7 years. Current treatment initiated at the age of forty included massage therapy, manual traction, ischemic pressure, and comprehensive manipulative medicine (CMM). A left-right chest circumference inequity was reduced by >10 cm, in correlation with improved appearance of the ribcage deformity and a 40% reduction in magnitude of Cobb angle, which had been stable for 30 years. The changes occurred gradually over an eight-year period, with the most rapid improvement occurring during two periods when CMM was employed.
Assuntos
Modalidades de Fisioterapia , Escoliose/reabilitação , Adolescente , Adulto , Fatores Etários , Criança , Pré-Escolar , Terapia Combinada , Terapia por Exercício , Feminino , Seguimentos , Humanos , Lactente , Recém-Nascido , Vértebras Lombares , Manipulação da Coluna , Massagem , Escoliose/classificação , Escoliose/diagnóstico , Vértebras TorácicasRESUMO
Root elongation occurs by the generation of new cells from meristematic tissue within the apical 1-2 mm region of root tips. Therefore penetration of the soil environment is carried out by newly synthesized plant tissue, whose cells are inherently vulnerable to invasion by pathogens. This conundrum, on its face, would seem to reflect an intolerable risk to the successful establishment of root systems needed for plant life. Yet root tip regions housing the meristematic tissues repeatedly have been found to be free of microbial infection and colonization. Even when spore germination, chemotaxis, and/or growth of pathogens are stimulated by signals from the root tip, the underlying root tissue can escape invasion. Recent insights into the functions of root border cells, and the regulation of their production by transient exposure to external signals, may shed light on long-standing observations.
Assuntos
Meristema/microbiologia , Meristema/fisiologia , Solo , Modelos BiológicosRESUMO
This review discusses how extracellular DNA (exDNA) might function in plant defense, and at what level(s) of innate immunity this process might operate. A new role for extracellular factors in mammalian defense has been described in a series of studies. These studies reveal that cells including neutrophils, eosinophils, and mast cells produce 'extracellular traps' (ETs) consisting of histone-linked exDNA. When pathogens are attracted to such ETs, they are trapped and killed. When the exDNA component of ETs is degraded, trapping is impaired and resistance against invasion is reduced. Conversely, mutation of microbial genes encoding exDNases that degrade exDNA results in loss of virulence. This discovery that exDNases are virulence factors opens new avenues for disease control. In plants, exDNA is required for defense of the root tip. Innate immunity-related proteins are among a group of >100 proteins secreted from the root cap and root border cell populations. Direct tests revealed that exDNA also is rapidly synthesized and exported from the root tip. When this exDNA is degraded by the endonuclease DNase 1, root tip resistance to fungal infection is lost; when the polymeric structure is degraded more slowly, by the exonuclease BAL31, loss of resistance to fungal infection is delayed accordingly. The results suggest that root border cells may function in a manner analogous to that which occurs in mammalian cells.
Assuntos
DNA de Plantas/imunologia , Imunidade Inata/genética , Meristema/microbiologia , Doenças das Plantas/imunologia , Imunidade Vegetal/genética , Plantas/imunologia , Animais , Bactérias/imunologia , Bactérias/patogenicidade , Sobrevivência Celular , DNA de Plantas/metabolismo , Desoxirribonuclease I/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/imunologia , Proteínas Fúngicas/metabolismo , Fungos/imunologia , Fungos/patogenicidade , Regulação da Expressão Gênica de Plantas , Mamíferos/genética , Mamíferos/imunologia , Meristema/citologia , Meristema/imunologia , Células Vegetais , Doenças das Plantas/microbiologia , Raízes de Plantas/citologia , Raízes de Plantas/imunologia , Raízes de Plantas/microbiologia , Plantas/microbiologia , Fatores de Tempo , Virulência , Fatores de Virulência/metabolismoAssuntos
Arabidopsis/crescimento & desenvolvimento , Flavonoides/fisiologia , Medicago sativa/crescimento & desenvolvimento , Transdução de Sinais/fisiologia , Arabidopsis/química , Arabidopsis/genética , Diferenciação Celular , Divisão Celular , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Flavonoides/análise , Medicago sativa/química , Medicago sativa/genética , Proteínas de Transporte de Monossacarídeos/deficiência , Proteínas de Transporte de Monossacarídeos/genética , Pigmentação , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , RNA Antissenso/genéticaRESUMO
BACKGROUND: Some patients with mild or moderate thoracic scoliosis (Cobb angle <50-60 degrees) suffer disproportionate impairment of pulmonary function associated with deformities in the sagittal plane and reduced flexibility of the spine and chest cage. Long-term improvement in the clinical signs and symptoms of childhood onset scoliosis in an adult, without surgical intervention, has not been documented previously. CASE PRESENTATION: A diagnosis of thoracic scoliosis (Cobb angle 45 degrees) with pectus excavatum and thoracic hypokyphosis in a female patient (DOB 9/17/52) was made in June 1964. Immediate spinal fusion was strongly recommended, but the patient elected a daily home exercise program taught during a 6-week period of training by a physical therapist. This regime was carried out through 1992, with daily aerobic exercise added in 1974. The Cobb angle of the primary thoracic curvature remained unchanged. Ongoing clinical symptoms included dyspnea at rest and recurrent respiratory infections. A period of multimodal treatment with clinical monitoring and treatment by an osteopathic physician was initiated when the patient was 40 years old. This included deep tissue massage (1992-1996); outpatient psychological therapy (1992-1993); a daily home exercise program focused on mobilization of the chest wall (1992-2005); and manipulative medicine (1994-1995, 1999-2000). Progressive improvement in chest wall excursion, increased thoracic kyphosis, and resolution of long-standing respiratory symptoms occurred concomitant with a >10 degree decrease in Cobb angle magnitude of the primary thoracic curvature. CONCLUSION: This report documents improved chest wall function and resolution of respiratory symptoms in response to nonsurgical approaches in an adult female, diagnosed at age eleven years with idiopathic scoliosis.
RESUMO
Plant defense involves a complex array of biochemical interactions, many of which occur in the extracellular environment. The apical 1- to 2-mm root tip housing apical and root cap meristems is resistant to infection by most pathogens, so growth and gravity sensing often proceed normally even when other sites on the root are invaded. The mechanism of this resistance is unknown but appears to involve a mucilaginous matrix or "slime" composed of proteins, polysaccharides, and detached living cells called "border cells." Here, we report that extracellular DNA (exDNA) is a component of root cap slime and that exDNA degradation during inoculation by a fungal pathogen results in loss of root tip resistance to infection. Most root tips (>95%) escape infection even when immersed in inoculum from the root-rotting pathogen Nectria haematococca. By contrast, 100% of inoculated root tips treated with DNase I developed necrosis. Treatment with BAL31, an exonuclease that digests DNA more slowly than DNase I, also resulted in increased root tip infection, but the onset of infection was delayed. Control root tips or fungal spores treated with nuclease alone exhibited normal morphology and growth. Pea (Pisum sativum) root tips incubated with [(32)P]dCTP during a 1-h period when no cell death occurs yielded root cap slime containing (32)P-labeled exDNA. Our results suggest that exDNA is a previously unrecognized component of plant defense, an observation that is in accordance with the recent discovery that exDNA from white blood cells plays a key role in the vertebrate immune response against microbial pathogens.
Assuntos
DNA de Plantas/metabolismo , Espaço Extracelular/metabolismo , Meristema/microbiologia , Nectria/fisiologia , Pisum sativum/metabolismo , Pisum sativum/microbiologia , Doenças das Plantas/microbiologia , Sequência de Bases , Sobrevivência Celular , Desoxirribonuclease I/metabolismo , Meristema/citologia , Meristema/metabolismo , Nectria/citologia , Pisum sativum/citologia , Fatores de TempoRESUMO
Mitosis and cell wall synthesis in the legume root cap meristem can be induced and synchronized by the nondestructive removal of border cells from the cap periphery. Newly synthesized cells can be examined microscopically as they differentiate progressively during cap development, and ultimately detach as a new population of border cells. This system was used to demonstrate that Pisum sativum L. fucosyl transferase (PsFut1) mRNA expression is strongly expressed in root meristematic tissues, and is induced >2-fold during a 5-h period when mitosis in the root cap meristem is increased. Expression of PsFut1 antisense mRNA in pea hairy roots under the control of the CaMV35S promoter, which exhibits meristem localized expression in pea root caps, resulted in a 50-60% reduction in meristem localized endogenous PsFut1 mRNA expression measured using whole mount in situ hybridization. Changes in gross levels of cell wall fucosylated xyloglucan were not detected, but altered surface localization patterns were detected using whole mount immunolocalization with CCRC-M1, an antibody that recognizes fucosylated xyloglucan. Emerging hairy roots expressing antisense PsFut1 mRNA appeared normal macroscopically but scanning electron microscopy of tissues with altered CCRC-M1 localization patterns revealed wrinkled, collapsed cell surfaces. As individual border cells separated from the cap periphery, cell death occurred in correlation with extrusion of cellular contents through breaks in the wall.
Assuntos
Regulação da Expressão Gênica de Plantas , Meristema/genética , Pisum sativum/genética , Raízes de Plantas/genética , Northern Blotting , Southern Blotting , Parede Celular/metabolismo , Parede Celular/ultraestrutura , DNA Antissenso/genética , Fucose/metabolismo , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Hibridização In Situ , Meristema/metabolismo , Meristema/ultraestrutura , Microscopia Eletrônica de Varredura , Pisum sativum/metabolismo , Pisum sativum/ultraestrutura , Coifa/genética , Coifa/metabolismo , Coifa/ultraestrutura , Raízes de Plantas/metabolismo , Raízes de Plantas/ultraestrutura , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Charles Darwin recognized the power of the root cap as a model for plant signalling and behavior, and used it to explore the ways plants sense and respond to diverse stimuli. Over ensuing decades, various groups have reported tantalizing clues regarding the role of a complex extracellular matrix that ensheaths the tip region housing the apical and root cap meristems. In the course of characterizing root tip resistance to infection and injury and the role border cells play in this phenomenon, we confirmed and extended early- and mid-20(th) century studies reporting enzyme activities secreted from the root cap. Multidimensional protein analysis revealed, in fact, that >100 proteins are actively synthesized and secreted from the root cap and border cells. This 'root cap secretome' appears to be a critical component of root tip resistance to infection. We have developed a microscopic assay to quantify the protein-based extracellular response to dynamic changes in environmental conditions including hydroponic culture, and present the results here. This tool provides a simple, direct measure that can be used to explore the ways border cells may function in the manner of white blood cells to trap, immobilize and neutralize threats to the growing root tip.