Abstract Hemicellulose and cellulose are essential polysaccharides for plant development and major components of cell wall. They are also an important energy source for the production of ethanol from plant biomass, but their conversion to fermentable sugars is hindered by the complex structure of cell walls. The glucuronic acid substitution of xylan (GUX) enzymes attach glucuronic acid to xylan, a major component of hemicellulose, decreasing the efficiency of enzymes used for ethanol production. Since loss-of-function gux mutants of Arabidopsis thaliana enhance enzyme accessibility and cell wall digestion without adverse phenotypes, GUX genes are potential targets for genetically improving energy crops. However, comprehensive identification of GUX in important species and their evolutionary history are largely lacking. Here, we identified putative GUX proteins using hidden Markov model searches with the GT8 domain and a GUX-specific motif, and inferred the phylogenetic relationship of 18 species with Maximum likelihood and Bayesian approaches. Each species presented a variable number of GUX, and their evolution can be explained by a mixture of divergent, concerted and birth-and-death evolutionary models. This is the first broad insight into the evolution of GUX gene family in plants and will potentially guide genetic and functional studies in species used for biofuel production.

Abstract The successful development of genetically engineered monocots using Agrobacterium-mediated transformation has created an increasing demand for compatible vectors. We have developed a new expression vector, pGVG, for efficient transformation and expression of different constructs for gene overexpression and silencing in sugarcane. The pCAMBIA2300 binary vector was modified by adding Gateway recombination sites for fast gene transfer between vectors and the maize polyubiquitin promoter Ubi-1 (ZmUbi1), which is known to drive high gene expression levels in monocots. Transformation efficiency using the pGVG vector reached up to 14 transgenic events per gram of transformed callus. Transgenic plants expressing the β-glucuronidase (GUS) reporter gene from pGVG showed high levels of GUS activity. qRT-PCR evaluations demonstrated success for both overexpression and hairpin-based silencing cassettes. Therefore, pGVG is suitable for plant transformation and subsequent applications for high-throughput production of stable transgenic sugarcane. The use of an expression cassette based on the ZmUbi1 promoter opens the possibility of using pGVG in other monocot species.

Stresses such as cold and drought can impair plant yield and induce a highly complex array of responses. Sugarcane (Saccharum spp.) is cultivated in tropical and subtropical areas and is considered a cold-sensitive plant. We previously showed that cold stress induces the expression of several genes in in vitro sugarcane plantlets. Here we characterize one of those genes, SsNAC23, a member of the NAC family of plant-specific transcription factors, which are induced by low temperature and other stresses in several plant species. The expression of SsNAC23 was induced in sugarcane plants exposed to low temperatures (4ºC). With the aim of further understanding the regulatory network in response to stress, we used the yeast two-hybrid system to identify sugarcane proteins that interact with SsNAC23. Using SsNAC23 as bait, we screened a cDNA expression library of sugarcane plants submitted to 4ºC for 48 h. Several interacting partners were identified, including stress-related proteins, increasing our knowledge on how sugarcane plants respond to cold stress. One of these interacting partners, a thioredoxin h1, offers insights into the regulation of SsNAC23 activity.

Os arranjos de cDNA são uma poderosa ferramenta para o estudo de padrões de expressão gênica. Os arranjos em membranas de náilon apresentam ainda a vantagem de poderem ser reutilizados diversas vezes. Porém, um ponto bastante delicado em estudos de expressão gênica em larga escala é a sensibilidade. No caso de arranjos em membranas de náilon, a detecção dos sinais pode ser afetada pelo envoltório plástico utilizado para manter as membranas úmidas. Nesse estudo, nós avaliamos os efeitos de cinco tipos de plásticos na transmissão radioativa detectada, no número de genes com sinal acima da emissão de fundo e na variabilidade dos dados. O plástico produzido com polietileno com 69 μm de espessura apresentou uma forte interferência na emissão radioativa, bloqueando 68.7% do sinal detectado. Este bloqueio na transmitância diminuiu o numero de genes detectados e aumentou a variabilidade dos dados. Outros plásticos mais finos tiveram resultados melhores. Apesar de plásticos feitos de cloreto de polivinilideno e cloreto de polivinila (ambos com 13 μm de espessura) e polietileno (29 e 7 μm de espessura) terem diferentes níveis de transmitância, todos apresentaram performances semelhantes nos testes realizados. Cloreto de polivinilideno e polietileno com 29 μm de espessura foram os plásticos escolhidos devido à facilidade de manuseio. Para outros tipos de plásticos, é recomendável realizar um teste de suas performances antes de utilizá-los para envolver membranas de náilon, de forma a obter o máximo de informação dos experimentos com arranjos de cDNA.

cDNA arrays are a powerful tool for discovering gene expression patterns. Nylon arrays have the advantage that they can be re-used several times. A key issue in high throughput gene expression analysis is sensitivity. In the case of nylon arrays, signal detection can be affected by the plastic bags used to keep membranes humid. In this study, we evaluated the effect of five types of plastics on the radioactive transmittance, number of genes with a signal above the background, and data variability. A polyethylene plastic bag 69 μm thick had a strong shielding effect that blocked 68.7% of the radioactive signal. The shielding effect on transmittance decreased the number of detected genes and increased the data variability. Other plastics which were thinner gave better results. Although plastics made from polyvinylidene chloride, polyvinyl chloride (both 13 μm thick) and polyethylene (29 and 7 μm thick) showed different levels of transmittance, they all gave similarly good performances. Polyvinylidene chloride and polyethylene 29 mm thick were the plastics of choice because of their easy handling. For other types of plastics, it is advisable to run a simple check on their performance in order to obtain the maximum information from nylon cDNA arrays.

O ácido jasmônico (JA) e seu éster metil-jasmonato (MeJA) são moléculas sinalizadoras derivadas do ácido linolênico e estão envolvidas no desenvolvimento da planta e na resposta aos estresses. MeJA regula a expressão gênica ao nível transcricional, do processamento do RNA e da tradução. Investigamos as mudanças na expressão gênica em folhas de cana-de-açúcar expostas ao MeJA usando arranjos de cDNA. O RNA total isolado a 0, 0,5, 1, 3, 6 e 12 horas após o tratamento com MeJA foi utilizado para a síntese de sondas contendo alfa-33P-dCTP, as quais foram, posteriormente, hibridizadas em membranas de náilon contendo 1.536 clones de cDNA. Um aumento significativo na expressão gênica em resposta ao MeJA foi detectado em genes que respondem a estresses e também em genes com função desconhecida, enquanto os genes que participam da fotossíntese e da assimilação de carboidrato foram reprimidos. A busca por domínios conservados em proteínas desconhecidas e a análise digital do perfil de expressão de mRNA revelaram possíveis proteínas novas relacionadas a estresses induzidas por MeJA e os tecidos onde os genes regulados por MeJA são preferivelmente expressos.

Jasmonic acid (JA) and its ester methyl jasmonate (MeJA) are linolenic acid-derived signaling molecules involved in plant development and stress responses. MeJA regulates gene expression at transcription, RNA processing and translation. We investigated the changes in gene expression in sugarcane leaves exposed to MeJA using cDNA arrays. Total RNA isolated at 0, 0.5, 1, 3, 6, and 12 h following MeJA treatment was labeled with alpha-33P-dCTP and hybridized to nylon filters containing 1,536 cDNA clones. A significant increase in gene expression in response to MeJA was detected for both novel and well known stress-related genes, while genes participating in photosynthesis and carbohydrate assimilation were down-regulated. Searches for conserved domains in unknown proteins and digital mRNA expression profile analysis revealed putative new stress-related proteins up-regulated by MeJA and the tissues where the MeJA-regulated genes are preferably expressed.

Várias espécies vegetais liberam ânions de ácidos orgânicos (AO) de suas raízes em resposta a íons tóxicos de alumínio (Al) presentes no ambiente radicular. Hipoteticamente esses AO complexam os íons de Al presentes no apoplasto da raiz e/ou na rizosfera evitando assim sua interação com componentes celulares e ainda sua penetração no simplasto da raiz. Dois padrões temporais de exsudação são reconhecidos. No padrão I, os AO são rapidamente liberados pelas raízes após a exposição das mesmas aos íons de Al. No padrão II de exsudação, ocorre um atraso na liberação de AO após exposição das raízes à solução contendo Al. Outros compostos além dos AO foram detectados em exsudatos radiculares e relacionados com mecanismos de resistência a Al em plantas. Espécies vegetais como trigo sarraceno e chá apresentam mecanismos de tolerância ao Al. Estes mecanismos conferem às plantas capacidade de inativar e de armazenar o Al em formas não tóxicas nas folhas. Os distúrbios induzidos por Al em rotas metabólicas ainda são desconhecidos, assim como a relação desses distúrbios com as mudanças nas concentrações de AO em raízes que estão sob estresse de Al. Altas concentrações internas de AO nas raízes nem sempre levam a altas taxas de exsudação desses compostos, mesmo quando a variabilidade espacial da concentração e da exsudação ao longo do eixo radicular é considerada. Certamente Al induz uma grande permeabilidade de AO em células jovens da raiz. Canais aniônicos localizados na membrana plasmática são os prováveis transportadores desses compostos orgânicos para fora da célula. Plantas que superexpressam genes envolvidos na síntese e na exsudação de AO bem como genes relacionados com a toxidez de Al foram desenvolvidas pela engenharia genética. Na maioria dos casos essas plantas tiveram uma maior capacidade para desenvolver sob estresse de Al. Esses resultados indicam, portanto, novas alternativas para o desenvolvimento de plantas mais adaptadas às condições de solos ácidos e com problemas de toxidez de Al. O impacto ambiental que a grande quantidade de exsudatos radiculares de plantas geneticamente modificadas pode causar, especialmente na microbiota do solo, é discutido.

Numerous plant species can release organic acid anions (OA) from their roots in response to toxic aluminium (Al) ions present in the rooting medium. Hypothetically OA complex Al in the root apoplast and/or rhizosphere and thus avoid its interaction with root cellular components and its entry in the root symplast. Two temporal patterns of root OA exudation are observed. In pattern I, OA release is rapidly activated after the contact of the root with Al ions while in pattern II there is a lag phase between the addition of Al and the beginning of OA release. Compounds other than OA have been detected in root exudates and are also correlated with Al resistance in plants. Plant species like buckwheat and tea show mechanisms of Al tolerance, which confer them the capacity to inactivate and store Al internally in the leaves. Disturbances in metabolic pathways induced by Al are still obscure and their relation to the altered OA concentration observed in roots under Al stress is not yet established. High concentrations of OA in roots do not always lead to high rates of OA release even when the spatial distribution of these two characteristics along the root axis is taken into account. Al induces high permeability to OA in young root cells and anion channels located in the cell membrane have been proposed to mediate the transport of OA to outside the cell. Genetically modified plants that overexpress genes involved in the biosynthesis and transport of OA as well as in Al toxicity events at the cell level have been generated. In most cases the transformations resulted in an improved ability of the plant to cope with Al stress. These promising findings reinforce the possibility of engineering plants with superior resistance to Al-toxic acid soils. The environmental impact of the large amounts of root exudates possibly conferred by these genetically modified plants is discussed, with special emphasis on soil microbiota.

The expressed sequence tags (ESTs) produced in the Forests project provide an invaluable opportunity to assess the Eucalyptus transcriptome. Besides providing information on the different proteins produced by this plant, it is possible to infer gene expression profiles because non-normalized cDNA libraries were used. The EST frequency from any gene is correlated to the transcript levels in the tissues from which the cDNA libraries were constructed. The goal of this work was to identify Eucalyptus genes that showed either differential expression pattern or were ubiquitously expressed in the tissues sampled in the Forests project. Six robust statistical tests and very restrictive rules were applied to gain confidence in the in silico data aiming to avoid false positives. Several genes with interesting expression profiles were identified and some of them were validated by RT-PCR.

Alumínio (Al) é um dos principais fatores que afetam o desenvolvimento de plantas em solos ácidos, reduzindo substancialmente a produtividade agrícola. Na América do Sul, cerca de 66% da superfície do solo apresenta acidez, onde a alta saturação de alumínio é uma das maiores limitações à prática agrícola. Apesar do crescente número de estudos, uma compreensão completa das bases bioquímicas e moleculares da tolerância ao alumínio em plantas está longe de ser alcançada. No caso da cana-de-açúcar, não há nada publicado sobre a regulação gênica induzida durante o stress por alumínio. O objetivo deste trabalho foi identificar genes de cana-de-açúcar relacionados com as várias vias metabólicas reconhecidamente envolvidas na resposta à toxidez do alumínio em outras espécies de plantas e leveduras. Para a maioria dos genes relacionados com alumínio em outras espécies foram identificados similares em cana-de-açúcar, tais como aqueles que codificam enzimas que combatem o stress oxidativo ou a infestação por patógenos, proteínas responsáveis pela exudação de ácidos orgânicos e pela transdução de sinais. O papel desses genes na tolerância ao alumínio é revisado. Devido ao alto grau de conservação do genoma entre espécies próximas de gramíneas como milho, cevada, sorgo e cana-de-açúcar, esses genes serão uma ferramenta valiosa para a melhor compreensão e manipulação da tolerância ao alumínio nestas espécies.

Aluminum is one of the major factors that affect plant development in acid soils, causing a substantial reduction in yield in many crops. In South America, about 66% of the land surface is made up of acid soils where high aluminum saturation is one of the main limiting factors for agriculture. The biochemical and molecular basis of aluminum tolerance in plants is far from being completely understood despite a growing number of studies, and in the specific case of sugarcane there are virtually no reports on the effects of gene regulation on aluminum stress. The objective of the work presented in this paper was to prospect the sugarcane expressed sequence tag (SUCEST) data bank for sugarcane genes related to several biochemical pathways known to be involved in the responses to aluminum toxicity in other plant species and yeast. Sugarcane genes similar to most of these genes were found, including those coding for enzymes that alleviate oxidative stress or combat infection by pathogens and those which code for proteins responsible for the release of organic acids and signal transducers. The role of these genes in aluminum tolerance mechanisms is reviewed. Due to the high level of genomic conservation in related grasses such as maize, barley, sorghum and sugarcane, these genes may be valuable tools which will help us to better understand and to manipulate aluminum tolerance in these species.