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1.
Plant Physiol ; 179(4): 1373-1385, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30593452

RESUMO

The basidiomycete Ustilago maydis causes smut disease in maize (Zea mays) by infecting all plant aerial tissues. The infection causes leaf chlorosis and stimulates the plant to produce nutrient-rich niches (i.e. tumors), where the fungus can proliferate and complete its life cycle. Previous studies have recorded high accumulation of soluble sugars and starch within these tumors. Using interdisciplinary approaches, we found that the sugar accumulation within tumors coincided with the differential expression of plant sugars will eventually be exported transporters and the proton/sucrose symporter Sucrose Transporter1 To accumulate plant sugars, the fungus deploys its own set of sugar transporters, generating a sugar gradient within the fungal cytosol, recorded by expressing a cytosolic glucose (Glc) Förster resonance energy transfer sensor. Our measurements indicated likely elevated Glc levels in hyphal tips during infection. Growing infected plants under dark conditions led to decreased plant sugar levels and loss of the fungal tip Glc gradient, supporting a tight link between fungal sugar acquisition and host supplies. Finally, the fungal infection causes a strong imbalance in plant sugar distribution, ultimately impacting seed set and yield.


Assuntos
Metabolismo dos Carboidratos , Interações Hospedeiro-Patógeno , Proteínas de Transporte de Monossacarídeos/metabolismo , Ustilago/metabolismo , Zea mays/microbiologia , Transferência Ressonante de Energia de Fluorescência , Sementes/crescimento & desenvolvimento , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismo
2.
New Phytol ; 218(2): 594-603, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29451311

RESUMO

Crop yield depends on efficient allocation of sucrose from leaves to seeds. In Arabidopsis, phloem loading is mediated by a combination of SWEET sucrose effluxers and subsequent uptake by SUT1/SUC2 sucrose/H+ symporters. ZmSUT1 is essential for carbon allocation in maize, but the relative contribution to apoplasmic phloem loading and retrieval of sucrose leaking from the translocation path is not known. Here we analysed the contribution of SWEETs to phloem loading in maize. We identified three leaf-expressed SWEET sucrose transporters as key components of apoplasmic phloem loading in Zea mays L. ZmSWEET13 paralogues (a, b, c) are among the most highly expressed genes in the leaf vasculature. Genome-edited triple knock-out mutants were severely stunted. Photosynthesis of mutants was impaired and leaves accumulated high levels of soluble sugars and starch. RNA-seq revealed profound transcriptional deregulation of genes associated with photosynthesis and carbohydrate metabolism. Genome-wide association study (GWAS) analyses may indicate that variability in ZmSWEET13s correlates with agronomical traits, especifically flowering time and leaf angle. This work provides support for cooperation of three ZmSWEET13s with ZmSUT1 in phloem loading in Z. mays.


Assuntos
Técnicas de Inativação de Genes , Proteínas de Membrana Transportadoras/metabolismo , Mutação/genética , Floema/metabolismo , Proteínas de Plantas/metabolismo , Zea mays/genética , Sequência de Bases , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana Transportadoras/genética , Filogenia , Proteínas de Plantas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Solubilidade , Amido/metabolismo
3.
Plant J ; 93(4): 675-685, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29160592

RESUMO

Plant breeders have developed crop plants that are resistant to pests, but the continual evolution of pathogens creates the need to iteratively develop new control strategies. Molecular tools have allowed us to gain deep insights into disease responses, allowing for more efficient, rational engineering of crops that are more robust or resistant to a greater number of pathogen variants. Here we describe the roles of SWEET and STP transporters, membrane proteins that mediate transport of sugars across the plasma membrane. We discuss how these transporters may enhance or restrict disease through controlling the level of nutrients provided to pathogens and whether the transporters play a role in sugar signaling for disease resistance. This review indicates open questions that require further research and proposes the use of genome editing technologies for engineering disease resistance.


Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Açúcares/metabolismo , Membrana Celular/metabolismo , Resistência à Doença/fisiologia , Proteínas de Plantas/genética , Plantas/metabolismo , Plantas/microbiologia , Transdução de Sinais , Simbiose
4.
Plant Cell Physiol ; 58(2): 298-306, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28007966

RESUMO

Symbiotic nitrogen fixation in legumes contributes greatly to the global nitrogen cycle on the earth. In nodules, resident rhizobia supply nitrogen nutrient fixed from atmospheric N2 to the host plant; in turn, the plant provides photosynthetic metabolites to bacteroids as a carbon source. In this process, various transporters are involved at different membrane systems; however, little is known at the molecular level about the flow of carbon from the host cells to the symbiotic bacteria. We have been studying transporters functioning in nodules of Lotus japonicus, and found that out of 13 SWEET genes in the L. japonicus genome LjSWEET3, a member of the SWEET transporter family, is highly expressed in nodules. The SWEET family was first identified in Arabidopsis, where members of the family are involved in phloem loading, nectar secretion, pollen nutrition and seed filling. The expression of LjSWEET3 strongly increased during nodule development and reached the highest level in mature nodules. Histochemical analysis using L. japonicus plants transformed with LjSWEET3 promoter:GUS (ß-glucuronidase) showed strong expression in the vascular systems of nodules. Analysis of an LjSWEET3-green fluorescent protein (GFP) fusion expressed in Nicotiana banthamiana and Coptis japonica indicates that LjSWEET3 localizes to the plasma membrane. Together these data are consistent with a role for LjSWEET3 in sugar translocation towards nodules and also suggest the possible existence of multiple routes of carbon supply into nodules.


Assuntos
Lotus/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Lotus/genética , Fixação de Nitrogênio/genética , Fixação de Nitrogênio/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas/genética , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/metabolismo , Sacarose/metabolismo
5.
FASEB J ; 30(10): 3644-3654, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27411857

RESUMO

SWEETs represent a new class of sugar transporters first described in plants, animals, and humans and later in prokaryotes. Plant SWEETs play key roles in phloem loading, seed filling, and nectar secretion, whereas the role of archaeal, bacterial, and animal transporters remains elusive. Structural analyses show that eukaryotic SWEETs are composed of 2 triple-helix bundles (THBs) fused via an inversion linker helix, whereas prokaryotic SemiSWEETs contain only a single THB and require homodimerization to form transport pores. This study indicates that SWEETs retained sugar transport activity in all kingdoms of life, and that SemiSWEETs are likely their ancestral units. Fusion of oligomeric subunits into single polypeptides during evolution of eukaryotes is commonly found for transporters. Phylogenetic analyses indicate that THBs of eukaryotic SWEETs may not have evolved by tandem duplication of an open reading frame, but rather originated by fusion between an archaeal and a bacterial SemiSWEET, which potentially explains the asymmetry of eukaryotic SWEETs. Moreover, despite the ancient ancestry, SWEETs had not been identified in fungi or oomycetes. Here, we report the identification of SWEETs in oomycetes as well as SWEETs and a potential SemiSWEET in primitive fungi. BdSWEET1 and BdSWEET2 from Batrachochytrium dendrobatidis, a nonhyphal zoosporic fungus that causes global decline in amphibians, showed glucose and fructose transport activities.-Hu, Y.-B., Sosso, D., Qu, X.-Q., Chen, L.-Q., Ma, L., Chermak, D., Zhang, D.-C., Frommer, W. B. Phylogenetic evidence for a fusion of archaeal and bacterial SemiSWEETs to form eukaryotic SWEETs and identification of SWEET hexose transporters in the amphibian chytrid pathogen Batrachochytrium dendrobatidis.


Assuntos
Quitridiomicetos/patogenicidade , Eucariotos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Animais , Transporte Biológico , Quitridiomicetos/isolamento & purificação , Relação Estrutura-Atividade
6.
Plant Physiol ; 171(1): 554-65, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27021190

RESUMO

Optimization of nitrogen fixation by rhizobia in legumes is a key area of research for sustainable agriculture. Symbiotic nitrogen fixation (SNF) occurs in specialized organs called nodules and depends on a steady supply of carbon to both plant and bacterial cells. Here we report the functional characterization of a nodule-specific Suc transporter, MtSWEET11 from Medicago truncatula MtSWEET11 belongs to a clade of plant SWEET proteins that are capable of transporting Suc and play critical roles in pathogen susceptibility. When expressed in mammalian cells, MtSWEET11 transported sucrose (Suc) but not glucose (Glc). The MtSWEET11 gene was found to be expressed in infected root hair cells, and in the meristem, invasion zone, and vasculature of nodules. Expression of an MtSWEET11-GFP fusion protein in nodules resulted in green fluorescence associated with the plasma membrane of uninfected cells and infection thread and symbiosome membranes of infected cells. Two independent Tnt1-insertion sweet11 mutants were uncompromised in SNF Therefore, although MtSWEET11 appears to be involved in Suc distribution within nodules, it is not crucial for SNF, probably because other Suc transporters can fulfill its role(s).


Assuntos
Medicago truncatula/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/microbiologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Meristema/genética , Meristema/metabolismo , Mutação , Proteínas de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Rhizobium/fisiologia , Nódulos Radiculares de Plantas/metabolismo , Nódulos Radiculares de Plantas/microbiologia , Sacarose/metabolismo , Simbiose/fisiologia
7.
Nat Genet ; 47(12): 1489-93, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26523777

RESUMO

Carbohydrate import into seeds directly determines seed size and must have been increased through domestication. However, evidence of the domestication of sugar translocation and the identities of seed-filling transporters have been elusive. Maize ZmSWEET4c, as opposed to its sucrose-transporting homologs, mediates transepithelial hexose transport across the basal endosperm transfer layer (BETL), the entry point of nutrients into the seed, and shows signatures indicative of selection during domestication. Mutants of both maize ZmSWEET4c and its rice ortholog OsSWEET4 are defective in seed filling, indicating that a lack of hexose transport at the BETL impairs further transfer of sugars imported from the maternal phloem. In both maize and rice, SWEET4 was likely recruited during domestication to enhance sugar import into the endosperm.


Assuntos
Produtos Agrícolas/genética , Endosperma/metabolismo , Hexoses/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Sementes/metabolismo , Zea mays/metabolismo , Transporte Biológico , Regulação da Expressão Gênica de Plantas , Humanos , Mutação/genética , Oryza/genética , Oryza/crescimento & desenvolvimento , Proteínas de Plantas/genética , Sementes/genética , Sementes/crescimento & desenvolvimento , Zea mays/genética , Zea mays/crescimento & desenvolvimento
8.
Curr Opin Plant Biol ; 25: 53-62, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25988582

RESUMO

Three families of transporters have been identified as key players in intercellular transport of sugars: MSTs (monosaccharide transporters), SUTs (sucrose transporters) and SWEETs (hexose and sucrose transporters). MSTs and SUTs fall into the major facilitator superfamily; SWEETs constitute a structurally different class of transporters with only seven transmembrane spanning domains. The predicted topology of SWEETs is supported by crystal structures of bacterial homologs (SemiSWEETs). On average, angiosperm genomes contain ∼20 paralogs, most of which serve distinct physiological roles. In Arabidopsis, AtSWEET8 and 13 feed the pollen; SWEET11 and 12 provide sucrose to the SUTs for phloem loading; AtSWEET11, 12 and 15 have distinct roles in seed filling; AtSWEET16 and 17 are vacuolar hexose transporters; and SWEET9 is essential for nectar secretion. The remaining family members await characterization, and could play roles in the gametophyte as well as other important roles in sugar transport in the plant. In rice and cassava, and possibly other systems, sucrose transporting SWEETs play central roles in pathogen resistance. Notably, the human genome also contains a glucose transporting isoform. Further analysis promises new insights into mechanism and regulation of assimilate allocation and a new potential for increasing crop yield.


Assuntos
Proteínas de Plantas/metabolismo , Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Transporte Biológico , Metabolismo dos Carboidratos , Carboidratos , Citoplasma/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas de Plantas/genética , Plantas/genética
9.
Plant J ; 82(4): 632-43, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25824104

RESUMO

Bacterial blight of rice is caused by the γ-proteobacterium Xanthomonas oryzae pv. oryzae, which utilizes a group of type III TAL (transcription activator-like) effectors to induce host gene expression and condition host susceptibility. Five SWEET genes are functionally redundant to support bacterial disease, but only two were experimentally proven targets of natural TAL effectors. Here, we report the identification of the sucrose transporter gene OsSWEET13 as the disease-susceptibility gene for PthXo2 and the existence of cryptic recessive resistance to PthXo2-dependent X. oryzae pv. oryzae due to promoter variations of OsSWEET13 in japonica rice. PthXo2-containing strains induce OsSWEET13 in indica rice IR24 due to the presence of an unpredicted and undescribed effector binding site not present in the alleles in japonica rice Nipponbare and Kitaake. The specificity of effector-associated gene induction and disease susceptibility is attributable to a single nucleotide polymorphism (SNP), which is also found in a polymorphic allele of OsSWEET13 known as the recessive resistance gene xa25 from the rice cultivar Minghui 63. The mutation of OsSWEET13 with CRISPR/Cas9 technology further corroborates the requirement of OsSWEET13 expression for the state of PthXo2-dependent disease susceptibility to X. oryzae pv. oryzae. Gene profiling of a collection of 104 strains revealed OsSWEET13 induction by 42 isolates of X. oryzae pv. oryzae. Heterologous expression of OsSWEET13 in Nicotiana benthamiana leaf cells elevates sucrose concentrations in the apoplasm. The results corroborate a model whereby X. oryzae pv. oryzae enhances the release of sucrose from host cells in order to exploit the host resources.


Assuntos
Oryza/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Xanthomonas/genética , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Doenças das Plantas/genética , Proteínas de Plantas/genética , Polimorfismo de Nucleotídeo Único/genética , Xanthomonas/patogenicidade
10.
Plant Cell ; 27(3): 607-19, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25794936

RESUMO

Developing plant embryos depend on nutrition from maternal tissues via the seed coat and endosperm, but the mechanisms that supply nutrients to plant embryos have remained elusive. Sucrose, the major transport form of carbohydrate in plants, is delivered via the phloem to the maternal seed coat and then secreted from the seed coat to feed the embryo. Here, we show that seed filling in Arabidopsis thaliana requires the three sucrose transporters SWEET11, 12, and 15. SWEET11, 12, and 15 exhibit specific spatiotemporal expression patterns in developing seeds, but only a sweet11;12;15 triple mutant showed severe seed defects, which include retarded embryo development, reduced seed weight, and reduced starch and lipid content, causing a "wrinkled" seed phenotype. In sweet11;12;15 triple mutants, starch accumulated in the seed coat but not the embryo, implicating SWEET-mediated sucrose efflux in the transfer of sugars from seed coat to embryo. This cascade of sequentially expressed SWEETs provides the feeding pathway for the plant embryo, an important feature for yield potential.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriologia , Arabidopsis/metabolismo , Endosperma/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Fenômenos Fisiológicos da Nutrição , Animais , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Endosperma/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Modelos Biológicos , Mutação/genética , Fenômenos Fisiológicos da Nutrição/efeitos dos fármacos , Oócitos/metabolismo , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Fenótipo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Amido/metabolismo , Sacarose/metabolismo , Sacarose/farmacologia , Fatores de Tempo , Xenopus laevis
11.
Nature ; 508(7497): 546-9, 2014 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-24670640

RESUMO

Angiosperms developed floral nectaries that reward pollinating insects. Although nectar function and composition have been characterized, the mechanism of nectar secretion has remained unclear. Here we identify SWEET9 as a nectary-specific sugar transporter in three eudicot species: Arabidopsis thaliana, Brassica rapa (extrastaminal nectaries) and Nicotiana attenuata (gynoecial nectaries). We show that SWEET9 is essential for nectar production and can function as an efflux transporter. We also show that sucrose phosphate synthase genes, encoding key enzymes for sucrose biosynthesis, are highly expressed in nectaries and that their expression is also essential for nectar secretion. Together these data are consistent with a model in which sucrose is synthesized in the nectary parenchyma and subsequently secreted into the extracellular space via SWEET9, where sucrose is hydrolysed by an apoplasmic invertase to produce a mixture of sucrose, glucose and fructose. The recruitment of SWEET9 for sucrose export may have been a key innovation, and could have coincided with the evolution of core eudicots and contributed to the evolution of nectar secretion to reward pollinators.


Assuntos
Arabidopsis/metabolismo , Glucosiltransferases/metabolismo , Néctar de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Sacarose/metabolismo , Alquil e Aril Transferases/metabolismo , Animais , Arabidopsis/citologia , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Brassica rapa/anatomia & histologia , Brassica rapa/enzimologia , Brassica rapa/metabolismo , Metabolismo dos Carboidratos , Espaço Extracelular/metabolismo , Flores/fisiologia , Glucosiltransferases/genética , Células HEK293 , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Oócitos , Néctar de Plantas/biossíntese , Polinização , Transporte Proteico , Homologia de Sequência , Amido/metabolismo , Nicotiana/anatomia & histologia , Nicotiana/enzimologia , Nicotiana/metabolismo , Xenopus , beta-Frutofuranosidase/metabolismo
12.
Proc Natl Acad Sci U S A ; 111(4): E521-9, 2014 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-24474801

RESUMO

Citrus bacterial canker (CBC) disease occurs worldwide and incurs considerable costs both from control measures and yield losses. Bacteria that cause CBC require one of six known type III transcription activator-like (TAL) effector genes for the characteristic pustule formation at the site of infection. Here, we show that Xanthomonas citri subspecies citri strain Xcc306, with the type III TAL effector gene pthA4 or with the distinct yet biologically equivalent gene pthAw from strain XccA(w), induces two host genes, CsLOB1 and CsSWEET1, in a TAL effector-dependent manner. CsLOB1 is a member of the Lateral Organ Boundaries (LOB) gene family of transcription factors, and CsSWEET1 is a homolog of the SWEET sugar transporter and rice disease susceptibility gene. Both TAL effectors drive expression of CsLOB1 and CsSWEET1 promoter reporter gene fusions when coexpressed in citrus or Nicotiana benthamiana. Artificially designed TAL effectors directed to sequences in the CsLOB1 promoter region, but not the CsSWEET1 promoter, promoted pustule formation and higher bacterial leaf populations. Three additional distinct TAL effector genes, pthA*, pthB, and pthC, also direct pustule formation and expression of CsLOB1. Unlike pthA4 and pthAw, pthB and pthC do not promote the expression of CsSWEET1. CsLOB1 expression was associated with the expression of genes associated with cell expansion. The results indicate that CBC-inciting species of Xanthomonas exploit a single host disease susceptibility gene by altering the expression of an otherwise developmentally regulated gene using any one of a diverse set of TAL effector genes in the pathogen populations.


Assuntos
Infecções Bacterianas/genética , Citrus/microbiologia , Genes de Plantas , Predisposição Genética para Doença , Xanthomonas/patogenicidade , Infecções Bacterianas/microbiologia , Parede Celular
13.
Proc Natl Acad Sci U S A ; 110(39): E3685-94, 2013 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-24027245

RESUMO

Eukaryotic sugar transporters of the MFS and SWEET superfamilies consist of 12 and 7 α-helical transmembrane domains (TMs), respectively. Structural analyses indicate that MFS transporters evolved from a series of tandem duplications of an ancestral 3-TM unit. SWEETs are heptahelical proteins carrying a tandem repeat of 3-TM separated by a single TM. Here, we show that prokaryotes have ancestral SWEET homologs with only 3-TM and that the Bradyrhizobium japonicum SemiSWEET1, like Arabidopsis SWEET11, mediates sucrose transport. Eukaryotic SWEETs most likely evolved by internal duplication of the 3-TM, suggesting that SemiSWEETs form oligomers to create a functional pore. However, it remains elusive whether the 7-TM SWEETs are the functional unit or require oligomerization to form a pore sufficiently large to allow for sucrose passage. Split ubiquitin yeast two-hybrid and split GFP assays indicate that Arabidopsis SWEETs homo- and heterooligomerize. We examined mutant SWEET variants for negative dominance to test if oligomerization is necessary for function. Mutation of the conserved Y57 or G58 in SWEET1 led to loss of activity. Coexpression of the defective mutants with functional A. thaliana SWEET1 inhibited glucose transport, indicating that homooligomerization is necessary for function. Collectively, these data imply that the basic unit of SWEETs, similar to MFS sugar transporters, is a 3-TM unit and that a functional transporter contains at least four such domains. We hypothesize that the functional unit of the SWEET family of transporters possesses a structure resembling the 12-TM MFS structure, however, with a parallel orientation of the 3-TM unit.


Assuntos
Proteínas de Bactérias/metabolismo , Metabolismo dos Carboidratos , Proteínas de Membrana Transportadoras/metabolismo , Família Multigênica , Proteínas de Plantas/metabolismo , Multimerização Proteica , Sacarose/metabolismo , Aminoácidos/metabolismo , Arabidopsis/metabolismo , Proteínas de Bactérias/química , Transporte Biológico , Bradyrhizobium/metabolismo , Teste de Complementação Genética , Glucose/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Membrana Transportadoras/química , Modelos Biológicos , Filogenia , Proteínas de Plantas/química , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade
14.
Curr Opin Plant Biol ; 16(3): 389-95, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23587939

RESUMO

Revolutionary new technologies, namely in the areas of DNA sequencing and molecular imaging, continue to impact new discoveries in plant science and beyond. For decades we have been able to determine properties of enzymes, receptors and transporters in vitro or in heterologous systems, and more recently been able to analyze their regulation at the transcriptional level, to use GFP reporters for obtaining insights into cellular and subcellular localization, and tp measure ion and metabolite levels with unprecedented precision using mass spectrometry. However, we lack key information on the location and dynamics of the substrates of enzymes, receptors and transporters, and on the regulation of these proteins in their cellular environment. Such information can now be obtained by transitioning from in vitro to in vivo biochemistry using biosensors. Genetically encoded fluorescent protein-based sensors for ion and metabolite dynamics provide highly resolved spatial and temporal information, and are complemented by sensors for pH, redox, voltage, and tension. They serve as powerful tools for identifying missing processes (e.g., glucose transport across ER membranes), components (e.g., SWEET sugar transporters for cellular sugar efflux), and signaling networks (e.g., from systematic screening of mutants that affect sugar transport or cytosolic and vacuolar pH). Combined with the knowledge of properties of enzymes and transporters and their interactions with the regulatory machinery, biosensors promise to be key diagnostic tools for systems and synthetic biology.


Assuntos
Bioquímica/métodos , Técnicas Biossensoriais/métodos , Imagem Molecular/métodos , Fenômenos Fisiológicos Vegetais , Transporte Biológico , Proteínas de Transporte/metabolismo , Enzimas/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Glucose/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Concentração de Íons de Hidrogênio , Transdução de Sinais , Biologia Sintética/métodos
15.
J Exp Bot ; 63(16): 5843-57, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22945943

RESUMO

The pentatricopeptide repeat (PPR) domain is an RNA binding domain allowing members of the PPR superfamily to participate in post-transcriptional processing of organellar RNA. Loss of PPR8522 from maize (Zea mays) confers an embryo-specific (emb) phenotype. The emb8522 mutation was isolated in an active Mutator (Mu) population and co-segregation analysis revealed that it was tightly linked to a MuDR insertion in the first exon of PPR8522. Independent evidence that disruption of PPR8522 caused the emb phenotype was provided by fine mapping to a region of 116kb containing no other gene than PPR8522 and complementation of the emb8522 mutant by a PPR8522 cDNA. The deduced PPR8522 amino acid sequence of 832 amino acids contains 10 PPR repeats and a chloroplast target peptide, the function of which was experimentally demonstrated by transient expression in Nicotiana benthamiana. Whereas mutant endosperm is apparently normal, mutant embryos deviate from normal development as early as 3 days after pollination, are reduced in size, exhibit more or less severe morphological aberrations depending on the genetic background, and generally do not germinate. The emb8522 mutation is the first to associate the loss of a PPR gene with an embryo-lethal phenotype in maize. Analyses of mutant plantlets generated by embryo-rescue experiments indicate that emb8522 also affects vegetative plant growth and chloroplast development. The loss of chloroplast transcription dependent on plastid-encoded RNA polymerase is the likely cause for the lack of an organized thylakoid network and an albino, seedling-lethal phenotype.


Assuntos
Cloroplastos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismo , Sequência de Aminoácidos , Cloroplastos/química , Cloroplastos/genética , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Proteínas de Plantas/genética , Estrutura Terciária de Proteína , Transporte Proteico , Alinhamento de Sequência , Zea mays/embriologia , Zea mays/genética
16.
J Vis Exp ; (65)2012 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-22805296

RESUMO

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days. We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging(1) (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers(2). This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution. Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors(3).


Assuntos
Arabidopsis/crescimento & desenvolvimento , Transferência Ressonante de Energia de Fluorescência/métodos , Técnicas Analíticas Microfluídicas/métodos , Raízes de Plantas/crescimento & desenvolvimento , Imagem com Lapso de Tempo/métodos , Dimetilpolisiloxanos/química , Plântula/crescimento & desenvolvimento
17.
Plant Cell ; 24(2): 676-91, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22319053

RESUMO

RNA editing plays an important role in organelle gene expression in various organisms, including flowering plants, changing the nucleotide information at precise sites. Here, we present evidence that the maize (Zea mays) nuclear gene Pentatricopeptide repeat 2263 (PPR2263) encoding a DYW domain-containing PPR protein is required for RNA editing in the mitochondrial NADH dehydrogenase5 (nad5) and cytochrome b (cob) transcripts at the nad5-1550 and cob-908 sites, respectively. Its putative ortholog, MITOCHONDRIAL EDITING FACTOR29, fulfills the same role in Arabidopsis thaliana. Both the maize and the Arabidopsis proteins show preferential localization to mitochondria but are also detected in chloroplasts. In maize, the corresponding ppr2263 mutation causes growth defects in kernels and seedlings. Embryo and endosperm growth are reduced, leading to the production of small but viable kernels. Mutant plants have narrower and shorter leaves, exhibit a strong delay in flowering time, and generally do not reach sexual maturity. Whereas mutant chloroplasts do not have major defects, mutant mitochondria lack complex III and are characterized by a compromised ultrastructure, increased transcript levels, and the induction of alternative oxidase. The results suggest that mitochondrial RNA editing at the cob-908 site is necessary for mitochondrion biogenesis, cell division, and plant growth in maize.


Assuntos
Citocromos b/genética , Proteínas Mitocondriais/genética , NADH Desidrogenase/genética , Proteínas de Plantas/metabolismo , Edição de RNA , Zea mays/crescimento & desenvolvimento , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Cloroplastos/enzimologia , Regulação da Expressão Gênica de Plantas , Microscopia Eletrônica de Transmissão , Mitocôndrias/enzimologia , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Mutagênese Insercional , Oxirredutases/metabolismo , Fenótipo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , RNA de Plantas/genética , Sementes/crescimento & desenvolvimento , Zea mays/genética , Zea mays/metabolismo
18.
Science ; 335(6065): 207-11, 2012 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-22157085

RESUMO

Plants transport fixed carbon predominantly as sucrose, which is produced in mesophyll cells and imported into phloem cells for translocation throughout the plant. It is not known how sucrose migrates from sites of synthesis in the mesophyll to the phloem, or which cells mediate efflux into the apoplasm as a prerequisite for phloem loading by the SUT sucrose-H(+) (proton) cotransporters. Using optical sucrose sensors, we identified a subfamily of SWEET sucrose efflux transporters. AtSWEET11 and 12 localize to the plasma membrane of the phloem. Mutant plants carrying insertions in AtSWEET11 and 12 are defective in phloem loading, thus revealing a two-step mechanism of SWEET-mediated export from parenchyma cells feeding H(+)-coupled import into the sieve element-companion cell complex. We discuss how restriction of intercellular transport to the interface of adjacent phloem cells may be an effective mechanism to limit the availability of photosynthetic carbon in the leaf apoplasm in order to prevent pathogen infections.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Floema/metabolismo , Sacarose/metabolismo , Animais , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Transporte Biológico , Membrana Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas Mutantes/metabolismo , Oryza/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Regiões Promotoras Genéticas
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