O estresse salino afeta o crescimento e o desenvolvimento das plantas, induzindo respostas bioquímicas e fisiológicas como mecanismo de adaptação para sua sobrevivência. As proteínas desacopladoras de planta (pUCPs) dissipam o gradiente eletroquímico mitocondrial de prótons como calor e são codificadas por famílias multigênicas. Elas atuam como sistema de defesa evitando a formação de espécies reativas de oxigênio geradas em resposta a estresses ambientais. O objetivo do trabalho foi estudar a expressão de genes das pUCPs (VuUCP1a e VuUCP1b ) em raízes e folhas de plântulas de Vigna unguiculata submetidas ao estresse salino (NaCl 100 mM). Sementes foram germinadas no escuro e após 3 dias, as plântulas foram transferidas para solução de Hoagland, permanecendo 3 dias antes da aplicação do estresse. As raízes e folhas foram coletadas com 0; 6; 12 e 24 horas, após adição de NaCl, para avaliar o perfil de expressão dos genes da pUCP por RT- PCR. A análise de transcritos mostrou que VuUCP1a é expresso em raízes e folhas revelando um perfil constitutivo enquanto que VuUCP1b é dependente do tecido, isto é, expresso nas folhas, sem alteração em resposta ao estresse e induzido pelo estresse salino nas raízes. A peculiaridade da duplicação gênica de pUCP1 em caupi com perfil diferencial de expressão, sugere tanto um papel dessa enzima nos mecanismos de ajustamento ao estresse salino quanto promove essa espécie como um modelo atrativo para compreensão do papel da família multigênica da pUCP em plantas.

Salt stress affects growth and development of plants, inducing a variety of physiological and biochemical responses as an adaptation mechanism for survival. The plant uncoupling mitochondrial proteins (pUCPs) are able to dissipate the proton electrochemical gradient as heat and are encoded by a multigene family. They works as defence systems avoiding the formation of reactive oxygen species promoted by environmental stress. The aim of this work was to study gene expression of pUCPs (VuUCP1a and VuUCP1b) in roots and leaves from Vigna unguiculata seedlings under salt stress (100 mM NaCl). Seeds were germinated in the dark and after 3 days, the seedlings were transferred to Hoagland's medium and grown for 3 additional days before being submitted to the stress condition. Roots and leaves were harvested at 0; 6; 12 and 24 hours after addition of NaCl for total RNA isolation and RT-PCR assays. Expression analysis by RT-PCR showed that VuUCP1a is constitutive in leaves and roots while VuUCP1b is expressed as tissue-dependent presenting a constitutive profile in leaves and a differential one in roots from seedlings under salt stress. The uniqueness of pUCP1 gene duplication in cowpea with differential expression suggest a role of this enzyme in the adjustment of salt stress as well as promotes this species as an attractive model to understand the role of pUCP gene members in plants.

O objetivo deste trabalho foi caracterizar biológica e molecularmente três isolados de Sugarcane mosaic virus (SCMV) de lavouras de milho, analisá-los filogeneticamente e discriminar polimorfismos do genoma. Plantas com sintomas de mosaico e nanismo foram coletadas em lavouras de milho, no Estado de São Paulo e no Município de Rio Verde, GO, e seus extratos foliares foram inoculados em plantas indicadoras e submetidos à análise sorológica com antissoros contra o SCMV, contra o Maize dwarf mosaic virus (MDMV) e contra o Johnsongrass mosaic virus (JGMV). Mudas de sorgo 'Rio' e 'TX 2786' apresentaram sintomas de mosaico após a inoculação dos três isolados, e o DAS-ELISA confirmou a infecção pelo SCMV. O RNA total foi extraído e usado para amplificação por transcriptase reversa seguida de reação em cadeia de polimerase (RT-PCR). Fragmentos específicos foram amplificados, submetidos à análise por polimorfismo de comprimento de fragmento de restrição (RFLP) e sequenciados. Foi possível discriminar os genótipos de SCMV isolados de milho de outros isolados brasileiros do vírus. Alinhamentos múltiplos e análises dos perfis filogenéticos corroboram esses dados e mostram diversidade nas sequências de nucleotídeos que codificam para a proteína capsidial, o que explica o agrupamento separado desses isolados e sugere sua classificação como estirpes distintas, em lugar de simples isolados geográficos.

The objective of this work was to characterize biologically and molecularly three Sugarcane mosaic virus (SCMV) isolates from maize crops in Brazil, to analyze them phylogenetically and to discriminate genome polymorphisms. Plants with mosaic and stunting symptons were collected from maize crops in the state of São Paulo and in Rio Verde county, GO, Brazil; their foliar extracts were inoculated in indicator plants and subjected to serological analysis with antisera to SCMV, Maize dwarf mosaic virus (MDMV) and Johnsongrass mosaic virus (JGMV). Sorghum 'Rio' and 'TX 2786' seedlings showed mosaic symptons after inoculation of the three isolates, and DAS-ELISA confirmed infection by SCMV in these plants. Total RNA was extracted from the infected leaves and was used as template for reverse transcriptase polymerase chain reaction (RT-PCR). Specific fragments were amplified, subjected to restriction fragment length polymorphism (RFLP) analysis and sequenced. It was possible to discriminate the SCMV maize isolates from other Brazilian isolates of the virus. Multiple alignments and phylogenetic profile analyses corroborate these data and show diversity in the sequence of nucleotides that code for capsidal protein, what explains the distinct clustering of these isolates, suggesting that they should be ranked as distinct SCMV strains rather than geographical isolates.

Os dois principais vírus que infectam o milho no Brasil são o Sugarcane mosaic virus (SCMV) e o Maize rayado fino virus (MRFV), cujos principais vetores são o afídeo Rhopalosiphum maidis e a cigarrinha Dalbulus maidis, respectivamente. O MRFV é freqüentemente encontrado em infecções mistas com fitoplasmas e espiroplasmas, causando as doenças denominadas enfezamentos do milho. Em uma lavoura de milho próxima a Santo Antonio da Posse, SP, cercada por campos de cana-de-açúcar, foi encontrada alta incidência de plantas apresentando mosaico, riscas, nanismo e espigas com falhas no enchimento de grãos. Análises serológicas com anti-soros específicos detectaram a presença do SCMV e MRFV nessas plantas. A infecção pelo SCMV também foi confirmada por RT-PCR com primers específicos e análise de seqüências. Em observações de preparações contrastadas negativamente em TEM, partículas flexuosas (ca.770 nm) e isométricas (ca.30 nm) foram detectadas. Em cortes ultrafinos, inclusões citoplasmáticas, típicas de Potyviridae, foram observadas; não foi encontrada a presença de espiroplasmas nem de fitoplasmas. Esses resultados mostram que a infecção conjunta por SCMV e MRFV pode ser responsável pelos danos encontrados nessa lavoura.

The two main viruses infecting maize in Brazil are Sugarcane mosaic virus (SCMV) and Maize rayado fino virus (MRFV), whose main vectors are the aphid Rhopalosiphum maidis and the leafhopper Dalbulus maidis, respectively. MRFV is frequently found in mixed infections with phytoplasms and spiroplasms causing the so-called corn stunting diseases. In a maize commercial field close to Santo Antonio da Posse, São Paulo state, surrounded by sugarcane fields, it was found a high incidence of plants showing mosaic, striping, stunting and cobs with decreased number of grains. Serological analysis with specific antisera detected the presence of SCMV and MRFV in these plants. SCMV infection was also confirmed by RT-PCR with specific primers and sequence analysis. In TEM observations of negatively stained preparations, flexuous (ca.770 nm) and isometric particles (ca.30 nm) were detected. In ultrathin sections, cytoplasmic inclusions, typical of Potyviridae, were observed; neither presence of spiroplasms nor phytoplasms could be noted. These results show that mixed infection by SCMV and MRFV might be responsible for the crop losses.

The RNA interference (RNAi) technique is a recent technology that uses double-stranded RNA molecules to promote potent and specific gene silencing. The application of this technique to molecular biology has increased considerably, from gene function identification to disease treatment. However, not all small interfering RNAs (siRNAs) are equally efficient, making target selection an essential procedure. Here we present Strand Analysis (SA), a free online software tool able to identify and classify the best RNAi targets based on Gibbs free energy (deltaG). Furthermore, particular features of the software, such as the free energy landscape and deltaG gradient, may be used to shed light on RNA-induced silencing complex (RISC) activity and RNAi mechanisms, which makes the SA software a distinct and innovative tool.

Post-transcriptional gene silencing (PTGS) is a conserved surveillance mechanism that identifies and cleaves double-stranded RNA molecules and their cellular cognate transcripts. The RNA silencing response is actually used as a powerful technique (named RNA interference) for potent and specific inhibition of gene expression in several organisms. To identify gene products in Eucalyptus sharing similarities with enzymes involved in the PTGS pathway, we queried the expressed sequence tag database of the Brazilian Eucalyptus Genome Sequence Project Consortium (FORESTs) with the amino acid sequences of known PTGS-related proteins. Among twenty-six prospected genes, our search detected fifteen assembled sequences encoding products presenting high level of similarity (E value < 10-40) to proteins involved in PTGS in plants and other organisms. We conclude that most of the genes known to be involved in the PTGS pathway are represented in the FORESTs database.

Herbicides inhibit enzymatic systems of plants. Acetolactate synthase (ALS, EC = 4.1.3.18) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, EC 2.5.1.19) are key enzymes for herbicide action. Hundreds of compounds inhibit ALS. This enzyme is highly variable, enabling the selective control of weeds in a number of crops. Glyphosate, the only commercial herbicide inhibiting EPSPS is widely used for non-selective control of weeds in many crops. Recently, transgenic crops resistant to glyphosate were developed and have been used by farmers. The aim of this study was the data mining of eucalypt expressed sequence tags (ESTs) in the FORESTs Genome Project database (<A HREF="https://forests.esalq.usp.br">https://forests.esalq.usp.br</A>) related to these enzymes. Representative amino acid sequences from the NCBI database associated with ALS and EPSPS were blasted with ESTs from the FORESTs database using the tBLASTx option of the blast tool. The best blasting reads and clusters from FORESTs, represented as nucleotide sequences, were blasted back with the NCBI database to evaluate the level of similarity with available sequences from different species. One and seven clusters were identified as showing high similarity with EPSPS and ALS sequences from the literature, respectively. The alignment of EPSPS sequences allowed the identification of conserved regions that can be used to design specific primers for additional sequencings.

This work was aimed at locating Eucalyptus ESTs corresponding to the GS enzyme (Glutamine Synthetase, EC = 6.3.1.2) and to the D1 protein, which are directly related to resistance to herbicides that promote oxidative stress. Glutamine Synthetase corresponds to the site of action of the herbicide glufosinate. Herbicides that belong to groups such as ureas, uracils, triazines and triazinones act on the D1-Qb complex (receptor of electrons from the Photosystem II) by inactivating it. The clusters EGEQRT3302E01.g, EGEQRT3001F12.b; EGEZLV1203B04.g; EGBGFB1211H06.g and EGEZLV1205F09.g enclosed complete sequences (with 356 amino acids) of the Glutamine Synthetase enzyme. The cluster EGEQSL1054G06.g is a consensus of four reads and enclosed a complete sequence of D1 Protein (with 353 amino acids). The comparison of the sequences of Protein D1 from different species showed that the substitutions of serine (S) by glycine (G) or serine (S) by threonine (T) at the position 264 could produce plants resistant to herbicides that act on electron flow on Photosystem II. The sequence of amino acids corresponding to the cluster EGEQSL1054G06.g had a serine in position 264 indicating sensitivity of the Eucalyptus plants to herbicides that act on this site.

This work was aimed at locating Eucalyptus ESTs corresponding to the PROTOX or PPO enzyme (Protoporphyrinogen IX oxidase, E.C. 1.3.3.4) directly related to resistance to herbicides that promote oxidative stress, changing the functionality of this enzyme. PROTOX, which is the site of action of diphenyl-ether (oxyfluorfen, lactofen, fomesafen), oxadiazole (oxadiazon and oxadiargyl), and aryl triazolinone (sulfentrazone and carfentrazone) herbicides, acts on the synthesis route of porphyrins which is associated with the production of chlorophyll a, catalases, and peroxidases. One cluster and one single read were located, with e-values better than e-70, associated to PROTOX. The alignment results between amino acid sequences indicated that this enzyme is adequately represented in the ESTs database of the FORESTs project.