Proteomic and Transcriptomic Analyses Indicate Metabolic Changes and Reduced Defense Responses in Mycorrhizal Roots of Oeceoclades maculata (Orchidaceae) Collected in Nature.

Orchids form endomycorrhizal associations with fungi mainly belonging to basidiomycetes. The molecular events taking place in orchid mycorrhiza are poorly understood, although the cellular changes necessary to accommodate the fungus and to control nutrient exchanges imply a modulation of gene expression. Here, we used proteomics and transcriptomics to identify changes in the steady-state levels of proteins and transcripts in the roots of the green terrestrial orchid Oeceoclades maculata. When mycorrhizal and non-mycorrhizal roots from the same individuals were compared, 94 proteins showed differential accumulation using the label-free protein quantitation approach, 86 using isobaric tagging and 60 using 2D-differential electrophoresis. After de novo assembly of transcriptomic data, 11,179 plant transcripts were found to be differentially expressed, and 2175 were successfully annotated. The annotated plant transcripts allowed the identification of up- and down-regulated metabolic pathways. Overall, proteomics and transcriptomics revealed, in mycorrhizal roots, increased levels of transcription factors and nutrient transporters, as well as ethylene-related proteins. The expression pattern of proteins and transcripts involved in plant defense responses suggested that plant defense was reduced in O. maculata mycorrhizal roots sampled in nature. These results expand our current knowledge towards a better understanding of the orchid mycorrhizal symbiosis in adult plants under natural conditions.

Bacterial community assemblages associated with the phyllosphere, dermosphere, and rhizosphere of tree species of the Atlantic forest are host taxon dependent.

Bacterial communities associated with tree canopies have been shown to be specific to their plant hosts, suggesting that plant species-specific traits may drive the selection of microbial species that comprise their microbiomes. To further examine the degree to which the plant taxa drive the assemblage of bacterial communities in specific plant microenvironments, we evaluated bacterial community structures associated with the phyllosphere, dermosphere, and rhizosphere of seven tree species representing three orders, four families and four genera of plants from a pristine Dense Ombrophilous Atlantic forest in Brazil, using a combination of PCR-DGGE of 16S rRNA genes and clone library sequencing. Results indicated that each plant species selected for distinct bacterial communities in the phyllosphere, dermosphere, and rhizosphere, and that the bacterial community structures are significantly related to the plant taxa, at the species, family, and order levels. Further characterization of the bacterial communities of the phyllosphere and dermosphere of the tree species showed that they were inhabited predominantly by species of Gammaproteobacteria, mostly related to Pseudomonas. In contrast, the rhizosphere bacterial communities showed greater species richness and evenness, and higher frequencies of Alphaproteobacteria and Acidobacteria Gp1. With individual tree species each selecting for their specific microbiomes, these findings greatly increase our estimates of the bacterial species richness in tropical forests and provoke questions concerning the ecological functions of the microbial communities that exist on different plant parts.

Effects of stimulation of copper bioleaching on microbial community in vineyard soil and copper mining waste.

Long-term copper application in vineyards and copper mining activities cause heavy metal pollution sites. Such sites need remediation to protect soil and water quality. Bioremediation of contaminated areas through bioleaching can help to remove copper ions from the contaminated soils. Thus, the aim of this work was to evaluate the effects of different treatments for copper bioleaching in two diverse copper-contaminated soils (a 40-year-old vineyard and a copper mining waste) and to evaluate the effect on microbial community by applying denaturing gradient gel electrophoresis (DGGE) of 16S ribosomal DNA amplicons and DNA sequence analysis. Several treatments with HCl, H(2)SO(4), and FeSO(4) were evaluated by stimulation of bioleaching of copper in the soils. Treatments and extractions using FeSO(4) and H(2)SO(4) mixture at 30°C displayed more copper leaching than extractions with deionized water at room temperature. Treatment with H(2)SO(4) supported bioleaching of as much as 120 mg kg(-1) of copper from vineyard soil after 115 days of incubation. DGGE analysis of the treatments revealed that some treatments caused greater diversity of microorganisms in the vineyard soil compared to the copper mining waste. Nucleotide Blast of PCR-amplified fragments of 16S rRNA gene bands from DGGE indicated the presence of Rhodobacter sp., Silicibacter sp., Bacillus sp., Paracoccus sp., Pediococcus sp., a Myxococcales, Clostridium sp., Thiomonas sp., a firmicute, Caulobacter vibrioides, Serratia sp., and an actinomycetales in vineyard soil. Contrarily, Sphingomonas was the predominant genus in copper mining waste in most treatments. Paracoccus sp. and Enterobacter sp. were also identified from DGGE bands of the copper mining waste. Paracoccus species is involved in the copper bioleaching by sulfur oxidation system, liberating the copper bounded in the soils and hence promoting copper bioremediation. Results indicate that stimulation of bioleaching with a combination of FeSO(4) and H(2)SO(4) promoted bioleaching in the soils and can be employed ex situ to remediate copper-impacted soils.

Expression of putative pathogenicity-related genes in Xylella fastidiosa grown at low and high cell density conditions in vitro.

Xylella fastidiosa is the causal agent of economically important plant diseases, including citrus variegated chlorosis and Pierce's disease. Hitherto, there has been no information on the molecular mechanisms controlling X. fastidiosa-plant interactions. To determine whether predicted open reading frames (ORFs) encoding putative pathogenicity-related factors were expressed by X. fastidiosa 9a5c cells grown at low (LCD) and high cell density (HCD) conditions in liquid modified PW medium, reverse Northern blot hybridization and reverse transcription-polymerase chain reaction (RT-PCR) experiments were performed. Our results indicated that ORFs XF2344, XF2369, XF1851 and XF0125, encoding putative Fur, GumC, a serine-protease and RsmA, respectively, were significantly suppressed at HCD conditions. In contrast, ORF XF1115, encoding putative RpfF, was significantly induced at HCD conditions. Expressions of ORFs XF2367, XF2362 and XF0290, encoding putative GumD, GumJ and RpfA, respectively, were detected only at HCD conditions, whereas expression of ORF XF0287, encoding putative RpfB was detected only at LCD conditions. Bioassays with an Agrobacterium traG::lacZ reporter system indicated that X. fastidiosa does not synthesize N-acyl-homoserine lactones, whereas bioassays with a diffusible signal factor (DSF)-responsive Xanthomonas campestris pv. campestris mutant indicate that X. fastidiosa synthesizes a molecule similar to DSF in modified PW medium. Our data also suggest that the synthesis of the DSF-like molecule and fastidian gum by X. fastidiosa is affected by cell density in vitro.

In silico characterization and expression analyses of sugarcane putative sucrose non-fermenting-1 (SNF1) related kinases

Quinases de proteínas relacionadas a SNF1 (SnRK) podem desempenhar um papel importante na regulaçäo da expressäo gênica em células vegetais. Essa família de proteínas regulatórias é representada pela quinase de proteínas SNF1 (sucrose non-fermenting-1) em Saccharomyces cerevisiae, AMPKs (quinase de proteínas ativadas por AMP) em células de mamíferos e SnRKs (quinase de proteínas relacionadas a SNF1) em células vegetaìs. A família de SnRKs foi reorganizada em três subfamílias de acordo com suas relações filogenéticas com base nas seqüências de aminoácidos das proteínas. Membros das subfamílias de SnRKs foram identificados em diversas plantas. Existem evidências mostrando que essa família de proteínas está envolvida em resposta a estresses (nutricional e ambiental), apesar de seu papel näo ser totalmente compreendido. Nesse trabalho nós identificamos 22 contíguos de ESTs (expressed sequence tags) de cana-de-açúcar codificando SnRKs putativas. O alinhamento das seqüências de aminoácidos das SnRKs putativas de cana-de-açúcar com seqüências de aminoácidos de SnRKs identificadas em outras plantas revelaram um domínio catalítico N-terminal altamente conservado. Além disso, nossos resultados indicaram que em cana-de-açúcar há pelo menos um membro de cada subfamília de SnRKs. Análìse do padräo de expressäo dos contíguos de EST codificando para SnRK putativas nas 26 bibliotecas selecionadas do banco de dados do Sucest, indicou que membros dessa família de quinases säo expressos por toda planta. Membros de cada subfamília näo apresentaram padrões de expressäo específicos, sugerindo que suas funções näo estäo correlacionadas com sua relaçäo filogenética, com base nas seqüências de aminoácidos da regiäo N-terminal.

In silico differential display of defense-related expressed sequence tags from sugarcane tissues infected with diazotrophic endophytes

Os padrões de expressäo de 277 contíguos de ESTs de cana-de-açúcar codificando proteínas possivelmente associadas ao sistema de defesa vegetal, e.g. quitinases,β-1,3-glucanases, fenilalanina amonia liases, chalcona sintases, chalcona isomerases, isoflavona redutases, glicoproteínas ricas em hidroxiprolina, glicoproteínas ricas em prolina, peroxidases, catalases, superóxido dismutases, fatores de transcriçäo similares a WRKY e proteínas envolvidas no controle de morte celular, foram avaliados utilizando o banco de dados do SUCEST. Proteínas WRKY putativas de cana-de-açúcar foram comparadas e suas relações filogenéticas determinadas. Análises de agrupamento hierárquico foram utilizadas para a identificaçäo de ESTs relacionadas à defesa vegetal com padrões de expressäo similares em bibliotecas de cDNA representativas. Visando identificar ESTs relacionadas à defesa vegetal com expressäo diferencial em tecidos de cana-de-açúcar infectados com Cluconacetobacter diazotrophicus ou Herbaspirillum rubrisubalbicans, 179 contíguos de ESTs possivelmente associados à defesa expressos em tecidos näo-infectados (folhas e raízes) e/ou tecidos infectados foram selecionados e agrupados por similaridade de seus perfis de expressäo. Alterações nos níveis de expressäo de 124 contíguos de ESTs relacionados à defesa expressos em tecidos näo-infectados foram avaliadas em tecidos infectados. Aproximadamente 42 por cento desses contíguos e ESTs näo apresentaram expressäo em tecidos infectados, enquanto 15 por cento e 3 por cento apresentaram supressäo de mais de duas vezes em tecidos infectados com G. diazotrophicus ou H. rubrisubalbicans, respectivamente. Aproximadamente 14 e 8 por cento dos contíguos de ESTs avaliados apresentaram induçäo superior a duas vezes em tecidos infectados com G. diazotrophicus ou H. rubrisubalbicans, respectivamente. A expressäo diferencial de agrupamentos de genes relacionados à defesa vegetal pode ser importante para o estabelecimento e interações compatíveis entre plantas e diazotróficos endofíticos. Os resultados sugerem que o agrupamento hierárquico pode ser utilizado em escala genômica para a identificaçäo de genes possivelmente envolvidos no controle de interações planta-microorganismos.