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The sensitivity of root cells and root processes to toxic aluminium ions (Al3+) varies along the root axis. This study was established to assess the sensitivity of nutrient uptake to Al along the main root axis of maize genotypes that differ in resistance to Al and to test whether citrate, an Al-complexing compound that is unevenly released along the root axis, can play a role in protecting the root from Al-impaired nutrient uptake. A divided-root-chamber technique was used to measure net fluxes of calcium (Ca2+), magnesium (Mg2+), and potassium (K+) along intact roots of two maize genotypes differing in resistance to Al. The accumulation of Al along their main root axis was also measured in short-term experiments. Results of these experiments were compared with those of a previous study, where citrate exudation had been measured along identical maize root axes. Aluminium affected nutrient uptake widely along the root with strong effects in the apical region, reducing total Ca2+ and Mg2+ uptake, but not K+ uptake. The negative effects of Al3+ were more pronounced in the Al sensitive genotype than in the resistant one. The former also accumulated more Al in its roots than the latter, but this differential accumulation was observed only in the apical part of the root. The spatial pattern of nutrient uptake, irrespective of Al treatment, did not match that of Al-stimulated citrate exudation. Based on the differential sensitivity of the root axis of the two maize genotypes and especially on the extent of the root zones where these differences are expressed, it is suggested that the less Al-disturbed nutrient uptake of a genotype is associated with its resistance to Al.

Soil salinity is a limiting factor to sugar cane crop development, although in general plants present variable mechanisms of tolerance to salinity stress. The molecular basis underlying these mechanisms can be inferred by using proteomic analysis. Thus, the objective of this work was to identify differentially expressed proteins in sugar cane plants submitted to salinity stress. For that, a greenhouse experiment was established with four sugar cane varieties and two salt conditions, 0 mM (control) and 200 mM NaCl. Physiological and proteomics analyses were performed after 2 and 72 h of stress induction by salt. Distinct physiological responses to salinity stress were observed in the varieties and linked to tolerance mechanisms. In proteomic analysis, the roots soluble protein fraction was extracted, quantified, and analyzed through bidimensional electrophoresis. Gel images analyses were done computationally, where in each contrast only one variable was considered (salinity condition or variety). Differential spots were excised, digested by trypsin, and identified via mass spectrometry. The tolerant variety RB867515 showed the highest accumulation of proteins involved in growth, development, carbohydrate and energy metabolism, reactive oxygen species metabolization, protein protection, and membrane stabilization after 2 h of stress. On the other hand, the presence of these proteins in the sensitive variety was verified only in stress treatment after 72 h. These data indicate that these stress responses pathways play a role in the tolerance to salinity in sugar cane, and their effectiveness for phenotypical tolerance depends on early stress detection and activation of the coding genes expression.

The presence of aluminum (Al) is one of the main factors limiting crop yield in Brazil and worldwide. Plant responses to Al are complex, and the use of techniques such as microarrays can facilitate their comprehension. In a previous work, we evaluated the transcriptome of two maize lines, Cat100-6 and S1587-17, after growing the plants for 1 or 3 days in acid soil (pH 4.1) or alkaline soil with Ca(OH)₂ (pH 5.5), and we identified genes that likely contribute to Al tolerance. The mapping of these genes to the chromosomes allowed the identification of the genes that are localized in maize QTLs previously reported in the literature as associated with the tolerant phenotype. We were able to map genes encoding proteins possibly involved with acid soil tolerance, such as the ones encoding an RNA binding protein, a protease inhibitor, replication factors, xyloglucan endotransglycosylase and cyclins, inside QTLs known to be important for the Al-tolerant phenotype.

Transcription mediated by RNA polymerase II depends on a set of different transcription factors to form the pre-initiation complex. TFIIA is involved in the construction of this complex and increases the affinity of TBP for the DNA union region in vitro. In this study, we characterized the ScTFIIAgamma gene, which encodes a homolog of the smaller subunit (gamma) of transcription factor TFIIA in sugarcane. RNA blot analysis showed that ScTFIIAgamma transcripts accumulate in all tissues evaluated, with higher levels in leaf roll and flowers. In situ hybridization showed that ScTFIIAgamma was expressed in different cells of the reproductive meristem. In sugarcane plantlets, methyl jasmonate and absicic acid treatments as well as phosphate starvation had no influence on ScTFIIAgamma transcript accumulation. The subcelullar localization assay demonstrates that ScTFIIAgamma protein is directed to the cell nucleus. The phylogenetic analysis, the expression in several tissues and under different treatments and the nuclear localization are in line with the putative role of ScTFIIAgamma as a subunit of basal transcription factor.

Due to the strategic importance of sugarcane to Brazil, FAPESP, the main Sao Paulo state research funding agency, launched in 2008 the FAPESP Bioenergy Research Program (BIOEN, http://bioenfapesp.org). BIOEN aims to generate new knowledge and human resources for the improvement of the sugarcane and ethanol industry. As part of the BIOEN program, a Workshop on Sugarcane Improvement was held on March 18th and 19th 2009 in Sao Paulo, Brazil. The aim of the workshop was to explore present and future challenges for sugarcane improvement and its use as a sustainable bioenergy and biomaterial feedstock. The workshop was divided in four sections that represent important challenges for sugarcane improvement: a) gene discovery and sugarcane genomics, b) transgenics and controlled transgene expression, c) sugarcane physiology (photosynthesis, sucrose metabolism, and drought) and d) breeding and statistical genetics. This report summarizes the roadmap for the improvement of sugarcane.

UDP-glucose pyrophosphorylase (UGPase) is found in all organisms and catalyses the formation of UDP-glucose. In sugarcane, UDP-glucose is a branch-point in the carbon channelling into other carbohydrates, such as sucrose and cellulose, which are the major factors for sugarcane productivity. In most plants, UGPase has been described to be enzymatically active in the monomeric form, while in human and yeast, homo-octamers represent the active form of the protein. Here, we present the crystal structure of UGPase from sugarcane (ScUGPase-1) at resolution of 2.0 Å. The crystals of ScUGPase-1 reveal the presence of two molecules in the asymmetric unit and the multi-angle light scattering analysis shows that ScUGPase-1 forms a mixture of species ranging from monomers to larger oligomers in solution, suggesting similarities with the orthologs from yeast and human.

TGA factors play a key role in plant defense by binding to the promoter region of defense genes, inducing expression. Salicylic acid (SA) induces the expression of the gene encoding NIMIN-1, which interacts with NPR1/NIM1, a key regulator of systemic acquired resistance. We investigated whether the TGA2-binding motif TGACG located upstream of the NIMIN-1 gene is necessary for SA induction of NIMIN-1 expression. A mutated version of the NIMIN-1 promoter was created by site-directed mutagenesis. We generated T-DNA constructs in which native NIMIN-1 and mutated promoters were fused to green fluorescent protein and beta-glucuronidase reporters. We produced transgenic Arabidopsis plants and observed NIMIN-1 promoter-driven green fluorescent protein expression in the roots, petiole and leaves. Constructs were agroinfiltrated into the leaves for transient quantitative assays of gene expression. Using quantitative real-time RT-PCR, we characterized the normal gene response to SA and compared it to the response of the mutant version of the NIMIN-1 promoter. Both the native NIMIN-1 construct and an endogenous copy of NIMIN-1 were induced by SA. However, the mutated promoter construct was much less sensitive to SA than the native NIMIN-1 promoter, indicating that this TGA2-binding motif is directly involved in the modulation of SA-induced NIMIN-1 expression in Arabidopsis.

Sucrose is the major product of photosynthesis in many higher plants. It is transported from the source tissue through the phloem to various sink tissues to support plant growth, development and reproduction. Knowledge on the signal transduction pathways involved in sucrose synthesis in mature leaves is limited. Using a microarray approach, we analyzed the expression profiles of 1920 sugarcane genes encoding signal transduction elements, transcription factors and stress-related proteins. We used individuals from a population segregating for sugar content and gene expression profiles were obtained from seven individuals with highest and seven with lowest sugar content. Surprisingly, from the 24 differentially expressed genes, 19 were more expressed in plants containing low-sugar content. Three of these genes encoded 14-3-3 like proteins, which have been found to reduce sucrose phosphate synthase (SPS) activity. Another encoded an SNF1-related protein similar to a protein kinase that phosphorylates SPS in vitro making it a target for the interaction with 14-3-3 proteins. The up-regulation of eight stress related genes in the lower sugar content plants supports a view that sugar levels modulate a complex signal transduction network that seems to involve responses that are related to stress. Evidence that hormone signaling is related to the sucrose content was also found. These data reinforced the usefulness of genomic approaches to uncover how sucrose metabolism can be regulated in sugarcane.

To investigate the molecular mechanisms of Al toxicity, cross-species cDNA array approach was employed to identify expressed sequence tags (ESTs) regulated by Al stress in root tips of Al-tolerant maize (Zea mays) genotype Cat100-6 and Al-sensitive genotype S1587-17. Due to the high degree of conservation observed between sugarcane and maize, we have analyzed the expression profiling of maize genes using 2 304 sugarcane (ESTs) obtained from different libraries. We have identified 85 ESTs in Al stressed maize root tips with significantly altered expression. Among the up-regulated ESTs, we have found genes encoding previously identified proteins induced by Al stress, such as phenyl ammonia-lyase, chitinase, Bowman-Birk proteinase inhibitor, and wali7. In addition, several novel genes up-and downregulated by Al stress were identified in both genotypes.

Summary • Aluminum (Al) toxicity is a major factor limiting crop yields on acid soils. There is considerable genotypic variation for Al tolerance in most common plant species. In maize (Zea mays), Al tolerance is a complex phenomenon involving multiple genes and physiological mechanisms yet uncharacterized. • To begin elucidating the molecular basis of maize Al toxicity and tolerance, a detailed temporal analysis of root gene expression under Al stress was performed using microarrays with Al-tolerant and Al-sensitive genotypes. • Al altered the expression of significantly more genes in the Al-sensitive genotype, presumably as a result of more severe Al toxicity. Nevertheless, several Al-regulated genes exhibited higher expression in the Al-tolerant genotype. Cell wall-related genes, as well as low phosphate-responsive genes, were found to be regulated by Al. In addition, the expression patterns of genes related to Al-activated citrate release indicated that in maize this mechanism is probably regulated by the expression level and/or function of the citrate transporter. • This study is the first comprehensive survey of global transcriptional regulation under Al stress. The results described here will help to further our understanding of how mechanisms of Al toxicity and tolerance in maize are regulated at the transcriptional level.

The phytotoxic effects of aluminum (Al) on root systems of crop plants constitute a major agricultural problem in many areas of the world. Root exudation of Al-chelating molecules such as low-molecular-weight organic acids has been shown to be an important mechanism of plant Al tolerance/resistance. Differences observed in the physiology and electrophysiology of root function for two maize genotypes with contrasting Al tolerance revealed an association between rates of Al-activated root organic acid release and Al tolerance. Using these genotypes, we cloned ZmALMT1, a maize gene homologous to the wheat ALMT1 and Arabidopsis AtALMT1 genes that have recently been described as encoding functional, Al-activated transporters that play a role in tolerance by mediating Al-activated organic acid exudation in roots. The ZmALMT1 cDNA encodes a 451 amino acid protein containing six transmembrane helices. Transient expression of a ZmALMT1::GFP chimera confirmed that the protein is targeted to the plant cell plasma membrane. We addressed whether ZmALMT1 might underlie the Al-resistance response (i.e. Al-activated citrate exudation) observed in the roots of the Al-tolerant genotype. The physiological, gene expression and functional data from this study confirm that ZmALMT1 is a plasma membrane transporter that is capable of mediating elective anion efflux and influx. However, gene expression data as well as biophysical transport characteristics obtained from Xenopus oocytes expressing ZmALMT1 indicate that this transporter is implicated in the selective transport of anions involved in mineral nutrition and ion homeostasis processes, rather than mediating a specific Al-activated citrate exudation response at the rhizosphere of maize roots.

There is a growing demand for renewable energy, and sugarcane is a promising bioenergy crop. In Brazil, the largest sugarcane producer in the world, sugarcane plantations are expanding into areas where severe droughts are common. Recent evidence has highlighted the role of miRNAs in regulating drought responses in several species, including sugarcane. This review summarizes the data from miRNA expression profiles observed in a wide array of experimental conditions using different sugarcane cultivars that differ in their tolerance to drought. We uncovered a complex regulation of sugarcane miRNAs in response to drought and discussed these data with the miRNA profiles observed in other plant species. The predicted miRNA targets revealed different transcription factors, proteins involved in tolerance to oxidative stress, cell modification, as well as hormone signaling. Some of these proteins might regulate sugarcane responses to drought, such as reduction of internode growth and shoot branching and increased leaf senescence. A better understanding on the regulatory network from miRNAs and their targets under drought stress has a great potential to contribute to sugarcane improvement, either as molecular markers as well as by using biotechnological approaches.

The influence of xanthan and/or KCl addition on the properties of heat-induced soy protein isolate (SPI) gels at pH 3.0 was studied. Changes in protein solubility and subunit composition as well as in the mechanical properties, microstructure and water holding capacity of the gels were determined. The effect of KCl addition on each biopolymer solution was also investigated. The results indicated that SPI–xanthan gels prepared without KCl were mainly stabilized by non-covalent (H-bonding and hydrophobic) and SS bond interactions, whereas in gels containing KCl, electrostatic interactions were also involved in maintaining the gel structure. The b-7S subunit was probably the fraction electrostatically linked to the xanthan. The different values found for the mechanical properties after the addition of xanthan and/or KCl, were associated with the coarseness of the gel. Xanthan and KCl probably showed a synergistic effect on the Young modulus because KCl induced a conformation transition of the xanthan molecules. r 2006 Elsevier Ltd. All rights reserved.

The influence of the acidification rate and the final pH on the properties of sodium caseinate gels (2–6%, w/v) prepared by acidification with glucono-δ-lactone (GDL) at 10 °C was investigated. The rheological properties of the systems were analysed under shear at incipient gelation and under uniaxial compression throughout the entire gelation process. The water holding capacity (WHC) of these gels and their amount of soluble protein in different media were also evaluated. Up to ∼50% of the total κ-casein content was soluble in gels with a higher amount of GDL, and this contributed to the high WHC observed under this condition. The acidification rate did not affect the rheological properties measured under shear during gelation, but the GDL/caseinate ratio used had a significant effect on the steady-state properties. Fast acidification resulted in lower Young's modulus and stress at fracture values, but higher residual stress values and relaxation times. In contrast, slow acidification produced a more interconnected network probably because of the extensive reorganization or rearrangement within the segments near the isoelectric point.

The correlation between organic acid anion release and Al content was examined in two maize ( Zea mays L.) inbred lines, Cat 100-6 (Al-resistant) and S 1587-17 (Al-sensitive) treated with anion channel antagonists and La 3+ , a cation channel blocker. In the intact roots of Al-resistant maize, the Al-induced excretion of citrate was inhibited by the anion channel antagonists niflumic acid, anthracene-9-carboxylic and ethacrinic acid. Citrate release in excised root apices was reduced by 60% in the presence of 15 μM niflumic acid, while the Al content increased by 42%. Nevertheless, Cat 100-6 accumulated less Al than S 1587-17 when the rate of citrate release was similar in both lines, indicating that other mechanisms of Al-resistance are operating in Cat 100-6. The presence of 60 μM La 3+ did not change the rate of citrate release, but the Al content in excised root apices of Al-resistant plants was reduced by 70%. These results suggest that the Al distributed uniformly in the roots does not contribute to citrate release and possibly the activity of anion channels is correlated with the free activities of extracellular Al 3+ close to anion channels.

Coffea arabica, one of the most important breeding species in the world, is susceptible to the leaf miner Leucoptera coffeella, which causes severe damage to coffee plantations. A closely related coffee species (Coffea racemosa) resistant to this insect was crossed with C. arabica and resulted in segregating progenies with resistance or susceptibility to coffee leaf miner. The aim of this work was to isolate and characterize the genes involved in coffee resistance to this pest. Subtracted cDNA libraries enriched in genes preferentially expressed in coffee plants resistant to L. coffeella were constructed. Approximately 1500 clones were spotted on nylon membranes and hybridized to cDNA probes derived from RNA samples from infestation experiments. Several genes were differentially expressed. We selected expressed ESTs with the most interesting expression profiles and confirmed the up-regulation of five of them (Class III Chitinase PR-8, signal peptidase complex subunit SPC25, photosystem gene psaH, a putative calcium exchanger similar to CAX9 and a homeotic gene BEL) by RNA blot. The possible functions of these genes in coffee resistance and coffee development, and a hypothetical defense mechanism against L. coffeela are discussed.

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. 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.

The basic leucine zipper (bZIP) proteins form a large family of transcriptional factors in plants and other eukaryotes. Plant bZIP transcriptional factors are divided into subfamilies and are involved in regulating a large range of physiological processes, from plant development to responses to biotic and abiotic stimuli. In this work, we cloned a novel bZIP of sugarcane into the pET3c vector and expressed the recombinant SCbZIP1 (66-170) protein in Escherichia coli BL21 (DE3) plysS. The recombinant protein was purified by heat-treatment and reversed phase chromatography. Northern blot analysis showed that SCbZIP1 was expressed early in development on day 4, but was not induced by abscisic acid (ABA) or exposure to cold. The identity of the recombinant protein was confirmed by mass spectrometry and CD spectroscopy showed an alpha-helical content of 33%. Electrophoretic mobility assays showed that SCbZIP1 (66-170) bound strongly to G-box, Hex and C-box DNA motifs. SCbZIP1 (66-170) was phosphorylated in vitro by a series of protein kinases and its DNA-binding affinity was strongly decreased after phosphorylation by CKII. SCbZIP1 (66-170) also underwent homo- and heterodimerization with truncated forms of the bZIP transcription factor Opaque 2 from Coix.

Tropical and subtropical plants are generally sensitive to cold and can show appreciable variation in their response to cold stress when exposed to low positive temperatures. Using nylon filter arrays, we analyzed the expression profile of 1,536 expressed sequence tags (ESTs) of sugarcane (Saccharum sp. cv SP80-3280) exposed to cold for 3 to 48 h. Thirty-four cold-inducible ESTs were identified, of which 20 were novel cold-responsive genes that had not previously been reported as being cold inducible, including cellulose synthase, ABI3-interacting protein 2, a negative transcription regulator, phosphate transporter, and others, as well as several unknown genes. In addition, 25 ESTs were identified as being down-regulated during cold exposure. Using a database of cold-regulated proteins reported for other plants, we searched for homologs in the sugarcane EST project database (SUCEST), which contains 263,000 ESTs. Thirty-three homologous putative cold-regulated proteins were identified in the SUCEST database. On the basis of the expression profiles of the cold-inducible genes and the data-mining results, we propose a molecular model for the sugarcane response to low temperature.

The relation between Al-toxicity and oxidative stress was studied for two inbred lines of maize (Zea mays L.), Cat100-6 (Al-tolerant) and S1587-17 (Al-sensitive). Peroxidase (PX), catalase (CAT) and superoxide dismutase (SOD) activities were determined in root tips of both lines, exposed to different Al(3+) concentrations and times of exposure. No increases were observed in CAT activities in either line, although SOD and PX were found to be 1.7 and 2.0 times greater than initial levels, respectively, in sensitive maize treated with 36 microM of Al(3+) for 48 h. The results indicate that Al(3+) induces the dose- and time dependent formation of reactive oxygen species (ROS) and subsequent protein oxidation in S1587-17, although not in Cat100-6. After exposure to 36 microM of Al(3+) for 48 h, the formation of 20+/-2 nmol of carbonyls per mg of protein was observed in S1587-17. The onset of protein oxidation took place after the drop of the relative root growth observed in the sensitive line, indicating that oxidative stress is not the primary cause of root growth inhibition. The presence of Al(3+) did not induce lipid peroxidation in either lines, contrasting with the observations in other species. These results, in conjunction with the data presented in the literature, indicate that oxidative stress caused by Al may harm several components of the cell, depending on the plant species. Moreover, Al(3+) treatment and oxidative stress in the sensitive maize line induced cell death in root tip cells, an event revealed by the high chromatin fragmentation detected by TUNEL analysis.

Background Aluminum (Al) toxicity is one of the most important yield-limiting factors of many crops worldwide. The primary symptom of Al toxicity syndrome is the inhibition of root growth leading to poor water and nutrient absorption. Al tolerance has been extensively studied using hydroponic experiments. However, unlike soil conditions, this method does not address all of the components that are necessary for proper root growth and development. In the present study, we grew two maize genotypes with contrasting tolerance to Al in soil containing toxic levels of Al and then compared their transcriptomic responses. Results When grown in acid soil containing toxic levels of Al, the Al-sensitive genotype (S1587-17) showed greater root growth inhibition, more Al accumulation and more callose deposition in root tips than did the tolerant genotype (Cat100-6). Transcriptome profiling showed a higher number of genes differentially expressed in S1587-17 grown in acid soil, probably due to secondary effects of Al toxicity. Genes involved in the biosynthesis of organic acids, which are frequently associated with an Al tolerance response, were not differentially regulated in both genotypes after acid soil exposure. However, genes related to the biosynthesis of auxin, ethylene and lignin were up-regulated in the Al-sensitive genotype, indicating that these pathways might be associated with root growth inhibition. By comparing the two maize lines, we were able to discover genes up-regulated only in the Al-tolerant line that also presented higher absolute levels than those observed in the Al-sensitive line. These genes encoded a lipase hydrolase, a retinol dehydrogenase, a glycine-rich protein, a member of the WRKY transcriptional family and two unknown proteins. Conclusions This work provides the first characterization of the physiological and transcriptional responses of maize roots when grown in acid soil containing toxic levels of Al. The transcriptome profiles highlighted several pathways that are related to Al toxicity and tolerance during growth in acid soil. We found several genes that were not found in previous studies using hydroponic experiments, increasing our understanding of plant responses to acid soil. The use of two germplasms with markedly different Al tolerances allowed the identification of genes that are a valuable tool for assessing the mechanisms of Al tolerance in maize in acid soil.

Sugarcane is a monocot plant that accumulates sucrose to levels of up to 50% of dry weight in the stalk. The mechanisms that are involved in sucrose accumulation in sugarcane are not well understood, and little is known with regard to factors that control the extent of sucrose storage in the stalks. UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) is an enzyme that produces UDP-glucose, a key precursor for sucrose metabolism and cell wall biosynthesis. The objective of this work was to gain insights into the ScUGPase-1 expression pattern and regulatory mechanisms that control protein activity. ScUGPase-1 expression was negatively correlated with the sucrose content in the internodes during development, and only slight differences in the expression patterns were observed between two cultivars that differ in sucrose content. The intracellular localization of ScUGPase-1 indicated partial membrane association of this soluble protein in both the leaves and internodes. Using a phospho-specific antibody, we observed that ScUGPase-1 was phosphorylated in vivo at the Ser-419 site in the soluble and membrane fractions from the leaves but not from the internodes. The purified recombinant enzyme was kinetically characterized in the direction of UDP-glucose formation, and the enzyme activity was affected by redox modification. Preincubation with H2O2 strongly inhibited this activity, which could be reversed by DTT. Small angle x-ray scattering analysis indicated that the dimer interface is located at the C terminus and provided the first structural model of the dimer of sugarcane UGPase in solution. Fil: Soares, Jose Sergio M.. Universidade Estadual de Campinas. Instituto de Biologia. Departamento de Genética, Evolução e Bioagentes; Brasil Fil: Gentile, Agustina. Universidade Estadual de Campinas. Instituto de Biologia. Departamento de Genética, Evolução e Bioagentes; Brasil Fil: Scorsato, Valeria. Universidade Estadual de Campinas. Instituto de Química. Laboratório de Biologia Estrutural e Cristalografia; Brasil Fil: Lima, Aline da C.. Universidade Estadual de Campinas. Instituto de Química. Laboratório de Biologia Estrutural e Cristalografia; Brasil Fil: Kiyota, Eduardo. Universidade Estadual de Campinas. Instituto de Química. Laboratório de Biologia Estrutural e Cristalografia; Brasil Fil: Santos, Marcelo Leite Dos . Universidade Federal do Sergipe. Centro de Ciências Exatas e Tecnologia. Núcleo de Química; Brasil Fil: Piattoni, Claudia Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina. Universidad Nacional del Litoral; Argentina Fil: Huber, Steven C.. University of Illinois at Urbana-Champaign. Department of Agriculture Agricultural Research Service, and Department of Plant Biology; Estados Unidos Fil: Aparicio, Ricardo. Universidade Estadual de Campinas. Instituto de Química. Laboratório de Biologia Estrutural e Cristalografia; Brasil Fil: Menossi, Marcelo. Universidade Estadual de Campinas. Instituto de Biologia. Departamento de Genética, Evolução e Bioagentes; Brasil

Soil acidity limits crop yields worldwide and is a common result of aluminum (Al) phytotoxicity, which is known to inhibit root growth. Here, we compared the transcriptome of leaves from maize seedlings grown under control conditions (soil without free Al) and under acidic soil containing toxic levels of Al. This study reports, for the first time, the complex transcriptional changes that occur in the leaves of maize plants grown in acidic soil with phytotoxic levels of Al. Our data indicate that 668 genes were differentially expressed in the leaves of plants grown in acidic soil, which is significantly greater than that observed in our previous work with roots. Genes encoding TCA cycle enzymes were upregulated, although no specific transporter of organic acids was differentially expressed in leaves. We also provide evidence for positive roles for auxin and brassinosteroids in Al tolerance, whereas gibberellin and jasmonate may have negative roles. Our data indicate that plant responses to acidic soil with high Al content are not restricted to the root; tolerance mechanisms are also displayed in the aerial parts of the plant, thus indicating that the entire plant responds to stress.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T19:04:49Z No. of bitstreams: 1 S1415-47572001000100029.pdf: 471422 bytes, checksum: 9f6d2c612cfece31b16279c77fe02e2e (MD5) Made available in DSpace on 2013-08-22T19:04:49Z (GMT). No. of bitstreams: 1 S1415-47572001000100029.pdf: 471422 bytes, checksum: 9f6d2c612cfece31b16279c77fe02e2e (MD5) Previous issue date: 2001-12-01 Made available in DSpace on 2013-09-30T20:08:10Z (GMT). No. of bitstreams: 2 S1415-47572001000100029.pdf: 471422 bytes, checksum: 9f6d2c612cfece31b16279c77fe02e2e (MD5) S1415-47572001000100029.pdf.txt: 53325 bytes, checksum: 6b93110fb9696b5522176d7c349bbe04 (MD5) Previous issue date: 2001-12-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T15:17:24Z No. of bitstreams: 2 S1415-47572001000100029.pdf: 471422 bytes, checksum: 9f6d2c612cfece31b16279c77fe02e2e (MD5) S1415-47572001000100029.pdf.txt: 53325 bytes, checksum: 6b93110fb9696b5522176d7c349bbe04 (MD5) Made available in DSpace on 2014-05-20T15:17:24Z (GMT). No. of bitstreams: 2 S1415-47572001000100029.pdf: 471422 bytes, checksum: 9f6d2c612cfece31b16279c77fe02e2e (MD5) S1415-47572001000100029.pdf.txt: 53325 bytes, checksum: 6b93110fb9696b5522176d7c349bbe04 (MD5) Previous issue date: 2001-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) 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. Universidade Estadual de Campinas Centro de Biologia Molecular e Engenharia Genética Laboratório de Genoma Funcional Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) Milho e Sorgo Núcleo de Biologia Aplicada Universidade Estadual Paulista Faculdade de Agricultura e Veterinária Departamento de Biologia Aplicada Universidade Estadual Paulista Faculdade de Agricultura e Veterinária Departamento de Biologia Aplicada

The role of calmodulin on Al toxicity was studied in two maize ( Zea mays L.) inbred lines, Cat 100-6 (Al-tolerant) and S 1587-17 (Al-sensitive). Increasing levels of Al induced the release of malate at similar rate by roots of both genotypes, while the exudation of citrate, a stronger Al-binding compound, was 3.5 times higher in Cat 100-6 seedlings exposed to 16.2×10 −6 Al 3+ activity. The calmodulin inhibitor trifluoperazine significantly reduced the root growth in both genotypes, mimicking the main effect of Al. However, when Cat 100-6 and S 1587-17 seedlings were challenged with Al in conjunction with trifluoperazine, no further reduction in root growth or any other effect of Al toxicity was observed. The rate of Al-induced citrate exudation by both genotypes was not affected by treatment with trifluoperazine or calmidazolium, another calmodulin inhibitor. The Al 3+ interaction with cytoplasmic CaM was estimated using models for the binding of Al 3+ and Mg 2+ with CaM and physiological concentrations of citrate, CaM, InsP 3 , ATP, ADP, Al 3+ and Mg 2+ . In this simulation, Al 3+ associated with citrate and InsP 3 , but not with CaM. We conclude that calmodulin is not relevant to the physiological processes leading to the Al tolerance in maize, nor is it a primary target for Al toxicity.

Maize aldose reductase (AR) is a member of the aldo-keto reductase superfamily. In contrast to human AR, maize AR seems to prefer the conversion of sorbitol into glucose. The apoenzyme was crystallized in space group P2(1)2(1)2(1), with unit-cell parameters a = 47.2, b = 54.5, c = 100.6 A and one molecule in the asymmetric unit. Synchrotron X-ray diffraction data were collected and a final resolution limit of 2.0 A was obtained after data reduction. Phasing was carried out by an automated molecular-replacement procedure and structural refinement is currently in progress. The refined structure is expected to shed light on the functional/enzymatic mechanism and the unusual activities of maize AR.

The translational construct, containing 719 bp of promoter and 5′-untranslated region and the first 16 bp of coding region, of the maize gene Hrgp encoding a hydroxyproline-rich glycoprotein fused to a glucuronidase reporter cassette, has been tested for activity in different maize tissues by microprojectile bombardment. The promoter has been found to be very active in the tissues of the plant, such as meristems or young shoots, with high cell wall formation activity where a high expression has also been shown for the endogenous gene. The promoter was also shown to be very active in cell types with a protection role such as in pericarp or styles and in cell types where the reinforcement of the cell wall is needed, as styles, auricles and cortical cells in the root tip. The promoter activity is developmentally regulated in the endosperm, being highest simultaneously with active cell division at the early-mid stages of development. In the presence of ethylene, the promoter shows an increased activity in accordance with the increment of mRNA accumulation observed in the plant upon ethylene treatment. It is concluded that the promoter fragment starting at −719 bp (numbering related to the ATG) of the Hrgp gene keeps the essential cis-DNA elements necessary for spatial, temporal and hormonal gene expression in maize.

Summary: This report describes an algorithm (intensity-dependent selection of expression ratios or ISER) developed to analyse DNA array data by optimizing the selection of genes with the most significant variations in expression amongst two RNA samples. The algorithm is designed for use when little or no replication of array hybridizations is available. Availability: ISER is written in R language, and its code and on-line version are freely available at https://ipe.cbmeg.unicamp.br/pub/PMmA Contact: menossi@unicamp.br Supplementary information: https://ipe.cbmeg.unicamp.br/pub/ISER

A protocol is described for simultaneous histochemical detection of LacZ and Gus activity in plant tissues after microprojectile bombardment. The suitability of two different Gus substrates (Salmon-glcA, Magenta-β-d-glcA) is compared. This detection system is used to assay the number of cells expressing either or both reporter gene. This technique is used as a qualitative assay to demonstrate the tissue specificity of a Hrgp promoter in maize embryos, and to measure ABA responsiveness of a Lea promoter in Arabidopsis. The promoter to be studied is linked to the gus reporter gene and the lacZ reporter gene linked to the CaMV 35S promoter is used as a constitutive internal control. The use of an internal control drastically reduces the data variation inherent to microprojectile bombardment.

Made available in DSpace on 2013-08-28T14:11:36Z (GMT). No. of bitstreams: 1 WOS000294678300009.pdf: 677555 bytes, checksum: ad618b66656cc8525114d2f7e9aaa25d (MD5) Made available in DSpace on 2013-09-30T18:37:55Z (GMT). No. of bitstreams: 2 WOS000294678300009.pdf: 677555 bytes, checksum: ad618b66656cc8525114d2f7e9aaa25d (MD5) WOS000294678300009.pdf.txt: 49587 bytes, checksum: 573fd52d922ca0fd3a6f37a99178f29e (MD5) Previous issue date: 2011-08-30 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:50:29Z No. of bitstreams: 2 WOS000294678300009.pdf: 677555 bytes, checksum: ad618b66656cc8525114d2f7e9aaa25d (MD5) WOS000294678300009.pdf.txt: 49587 bytes, checksum: 573fd52d922ca0fd3a6f37a99178f29e (MD5) Made available in DSpace on 2014-05-20T13:50:29Z (GMT). No. of bitstreams: 2 WOS000294678300009.pdf: 677555 bytes, checksum: ad618b66656cc8525114d2f7e9aaa25d (MD5) WOS000294678300009.pdf.txt: 49587 bytes, checksum: 573fd52d922ca0fd3a6f37a99178f29e (MD5) Previous issue date: 2011-08-30 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Financiadora de Estudos e Projetos (FINEP) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Background: Plants are challenged by a large number of environmental stresses that reduce productivity and even cause death. Both chloroplasts and mitochondria produce reactive oxygen species under normal conditions; however, stress causes an imbalance in these species that leads to deviations from normal cellular conditions and a variety of toxic effects. Mitochondria have uncoupling proteins (UCPs) that uncouple electron transport from ATP synthesis. There is evidence that UCPs play a role in alleviating stress caused by reactive oxygen species overproduction. However, direct evidence that UCPs protect plants from abiotic stress is lacking.Methodology/Principal Findings: Tolerances to salt and water deficit were analyzed in transgenic tobacco plants that overexpress a UCP (AtUCP1) from Arabidopsis thaliana. Seeds of AtUCP1 transgenic lines germinated faster, and adult plants showed better responses to drought and salt stress than wild-type (WT) plants. These phenotypes correlated with increased water retention and higher gas exchange parameters in transgenic plants that overexpress AtUCP1. WT plants exhibited increased respiration under stress, while transgenic plants were only slightly affected. Furthermore, the transgenic plants showed reduced accumulation of hydrogen peroxide in stressed leaves compared with WT plants.Conclusions/Significance: Higher levels of AtUCP1 improved tolerance to multiple abiotic stresses, and this protection was correlated with lower oxidative stress. Our data support previous assumptions that UCPs reduce the imbalance of reactive oxygen species. Our data also suggest that UCPs may play a role in stomatal closure, which agrees with other evidence of a direct relationship between these proteins and photosynthesis. Manipulation of the UCP protein expression in mitochondria is a new avenue for crop improvement and may lead to crops with greater tolerance for challenging environmental conditions. Univ Estadual Campinas, Inst Biol, Lab Genoma Func, Dept Genet Evolucao & Bioagentes, Campinas, Brazil Univ Estadual Paulista, Inst Biociencias, Dept Genet, Botucatu, SP, Brazil Univ Estadual Campinas, Inst Biol, Dept Biol Vegetal, Campinas, Brazil Univ Estadual Paulista, Inst Biociencias, Dept Genet, Botucatu, SP, Brazil

The characterization of a coffee gene encoding a protein similar to miraculin-like proteins, which are members of the plant Kunitz serine trypsin inhibitor (STI) family of proteinase inhibitors (PIs), is described. PIs are important proteins in plant defence against insects and in the regulation of proteolysis during plant development. This gene has high identity with the Richadella dulcifica taste-modifying protein miraculin and with the tomato protein LeMir; and was named as CoMir (Coffea miraculin). Structural protein modelling indicated that CoMir had structural similarities with the Kunitz STI proteins, but suggested specific folding structures. CoMir was up-regulated after coffee leaf miner (Leucoptera coffella) oviposition in resistant plants of a progeny derived from crosses between C. racemosa (resistant) and C. arabica (susceptible). Interestingly, this gene was down-regulated during coffee leaf miner herbivory in susceptible plants. CoMir expression was up-regulated after abscisic acid application and wounding stress and was prominent during the early stages of flower and fruit development. In situ hybridization revealed that CoMir transcripts accumulated in the anther tissues that display programmed cell death (tapetum, endothecium and stomium) and in the metaxylem vessels of the petals, stigma and leaves. In addition, the recombinant protein CoMir shows inhibitory activity against trypsin. According to the present results CoMir may act in proteolytic regulation during coffee development and in the defence against L. coffeella. The similarity of CoMir with other Kunitz STI proteins and the role of CoMir in plant development and plant stress are discussed.

Background The genus Bothrops is widespread throughout Central and South America and is the principal cause of snakebite in these regions. Transcriptomic and proteomic studies have examined the venom composition of several species in this genus, but many others remain to be studied. In this work, we used a transcriptomic approach to examine the venom gland genes of Bothrops alternatus, a clinically important species found in southeastern and southern Brazil, Uruguay, northern Argentina and eastern Paraguay. Results A cDNA library of 5,350 expressed sequence tags (ESTs) was produced and assembled into 838 contigs and 4512 singletons. BLAST searches of relevant databases showed 30% hits and 70% no-hits, with toxin-related transcripts accounting for 23% and 78% of the total transcripts and hits, respectively. Gene ontology analysis identified non-toxin genes related to general metabolism, transcription and translation, processing and sorting, (polypeptide) degradation, structural functions and cell regulation. The major groups of toxin transcripts identified were metalloproteinases (81%), bradykinin-potentiating peptides/C-type natriuretic peptides (8.8%), phospholipases A2 (5.6%), serine proteinases (1.9%) and C-type lectins (1.5%). Metalloproteinases were almost exclusively type PIII proteins, with few type PII and no type PI proteins. Phospholipases A2 were essentially acidic; no basic PLA2 were detected. Minor toxin transcripts were related to L-amino acid oxidase, cysteine-rich secretory proteins, dipeptidylpeptidase IV, hyaluronidase, three-finger toxins and ohanin. Two non-toxic proteins, thioredoxin and double-specificity phosphatase Dusp6, showed high sequence identity to similar proteins from other snakes. In addition to the above features, single-nucleotide polymorphisms, microsatellites, transposable elements and inverted repeats that could contribute to toxin diversity were observed. Conclusions Bothrops alternatus venom gland contains the major toxin classes described for other Bothrops venoms based on trancriptomic and proteomic studies. The predominance of type PIII metalloproteinases agrees with the well-known hemorrhagic activity of this venom, whereas the lower content of serine proteases and C-type lectins could contribute to less marked coagulopathy following envenoming by this species. The lack of basic PLA2 agrees with the lower myotoxicity of this venom compared to other Bothrops species with these toxins. Together, these results contribute to our understanding of the physiopathology of envenoming by this species.

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.

The present article reports on the characterization of ScBAK1, a leucine-rich repeat receptor-like kinase from sugarcane (Saccharum spp.), expressed predominantly in bundle-sheath cells of the mature leaf and potentially involved in cellular signaling cascades mediated by high levels of sugar in this organ. In this report, it was shown that the ScBAK1 sequence was similar to the brassinosteroid insensitive1-associated receptor kinase1 (BAK1). The putative cytoplasmatic domain of ScBAK1 contains all the amino acids characteristic of protein kinases, and the extracellular domain contains five leucine-rich repeats and a putative leucine zipper. Transcripts of ScBAK1 were almost undetectable in sugarcane roots or in any other sink tissue, but accumulated abundantly in the mature leaves. The ScBAK1 expression was higher in the higher sugar content individuals from a population segregating for sugar content throughout the growing season. In situ hybridization in sugarcane leaves showed that the ScBAK1 mRNA accumulated at much higher levels in bundle-sheath cells than in mesophyll cells. In addition, using biolistic bombardment of onion epidermal cells, it was shown that ScBAK1-GFP fusions were localized in the plasma membrane as predicted for a receptor kinase. All together, the present data indicate that ScBAK1 might be a receptor involved in the regulation of specific processes in bundle-sheath cells and in sucrose synthesis in mature sugarcane leaves.

The general transcription factor TFIIH is a multiprotein complex with different enzymatic activities such as helicase, protein kinase and DNA repair. MAT1 (menage a trois 1) is one of the TFIIH subunits that has kinase activity and it is the third subunit of the cyclin-dependent kinase (CDK)-activating kinase (CAK), CDK7- cyclin H. The main objective of this work was to characterize ScMAT1, a sugarcane gene encoding a MAT1 homolog. Northern blots and in situ hybridization results showed that ScMAT1 was expressed in sugarcane mature leaf, leaf roll and inflorescence, and it was not differentially expressed in any of the other tissues analyzed such us bud and roots. In addition, ScMAT1 was not differentially expressed during different stress conditions and treatment with hormones. In situ hybridization analyses also showed that ScMAT1 was expressed in different cell types during leaf development. In order to identify proteins that interact with ScMAT1, a yeast two hybrid assay with ScMAT1 as bait was used to screen a sugarcane leaf cDNA library. The screening of yeast two hybrids yielded 14 positive clones. One of them is a cytochrome p450 family protein involved in oxidative degradation of toxic compounds. Other clones isolated are also related to plant responses to stress. To determine the subcellular localization of ScMAT1, a ScMAT1-GFP fusion was assayed in onion epidermal cell and the fluorescence was localized to the nucleus, in agreement with the putative role of ScMAT1 as a basal transcription factor.

Reproductive failures are still common grounds for complaint by commercial swine producers. Porcine parvovirus (PPV) is associated with different clinical reproductive signs. The aim of the present study was to investigate PPV fetal infection at swine farms having ongoing reproductive performance problems. The presence of virus in fetal tissues was determined by nested-polymerase chain reaction assay directed to the conserved NS1 gene of PPV in aborted fetuses, mummies and stillborns. Fetuses show a high frequency of PPV infection (96.4%; n = 28). In 60.7% of the fetuses, PPV were detected in all tissue samples (lung, heart, thymus, kidney, and spleen). Viral infection differed among fetal tissues, with a higher frequency in the lung and heart (p0.05). Fetuses with up to 99 days of gestational age and from younger sows showed a higher frequency of PPV (p0.05). No significant difference in the presence of PPV was detected among the three clinical presentations. The results suggest that PPV remains an important pathogenic agent associated with porcine fetal death.

Because of the economical relevance of sugarcane and its high potential as a source of biofuel, it is important to understand how this crop will respond to the foreseen increase in atmospheric [CO(2)]. The effects of increased [CO(2)] on photosynthesis, development and carbohydrate metabolism were studied in sugarcane (Saccharum ssp.). Plants were grown at ambient (approximately 370 ppm) and elevated (approximately 720 ppm) [CO(2)] during 50 weeks in open-top chambers. The plants grown under elevated CO(2) showed, at the end of such period, an increase of about 30% in photosynthesis and 17% in height, and accumulated 40% more biomass in comparison with the plants grown at ambient [CO(2)]. These plants also had lower stomatal conductance and transpiration rates (-37 and -32%, respectively), and higher water-use efficiency (c.a. 62%). cDNA microarray analyses revealed a differential expression of 35 genes on the leaves (14 repressed and 22 induced) by elevated CO(2). The latter are mainly related to photosynthesis and development. Industrial productivity analysis showed an increase of about 29% in sucrose content. These data suggest that sugarcane crops increase productivity in higher [CO(2)], and that this might be related, as previously observed for maize and sorghum, to transient drought stress.

Sugarcane is an important tropical crop having C4 carbohydrate metabolism which, allied with its perennial nature, makes it one of the most productive cultivated plants. It is mostly used to produce sugar, accounting for almost two thirds of world production. Recently it has gained increased attention because of its important potential for bio-fuel production. However, sugarcane has one of the more complex crop genomes, which has long hampered the development of sugarcane genetics to support breeding for crop improvement programs. Sugarcane belongs to the genus Saccharum L, part of the Poaceae familly (Grasses) and the Andropogonae tribe, which encompasses only polyploid species.With the advent of molecular genomics, the sugarcane genome has become less mysterious, although its complexity has been confirmed in many aspects. Shortcuts to genomic analyses have been identified thanks to synteny conservation with other grasses, in particular sorghum and rice. Over time, new tools have become available for understanding the molecular bases behind sugarcane productivity and a renewed interest has surfaced in its genetics and physiology.

The phytotoxic effects of aluminum (Al) on root systems of crop plants constitute a major agricultural problem in many areas of the world. Root exudation of Al-chelating molecules such as low-molecular-weight organic acids has been shown to be an important mechanism of plant Al tolerance/resistance. Differences observed in the physiology and electrophysiology of root function for two maize genotypes with contrasting Al tolerance revealed an association between rates of Al-activated root organic acid release and Al tolerance. Using these genotypes, we cloned ZmALMT1, a maize gene homologous to the wheat ALMT1 and Arabidopsis AtALMT1 genes that have recently been described as encoding functional, Al-activated transporters that play a role in tolerance by mediating Al-activated organic acid exudation in roots. The ZmALMT1 cDNA encodes a 451 amino acid protein containing six transmembrane helices. Transient expression of a ZmALMT1::GFP chimera confirmed that the protein is targeted to the plant cell plasma membrane. We addressed whether ZmALMT1 might underlie the Al-resistance response (i.e. Al-activated citrate exudation) observed in the roots of the Al-tolerant genotype. The physiological, gene expression and functional data from this study confirm that ZmALMT1 is a plasma membrane transporter that is capable of mediating elective anion efflux and influx. However, gene expression data as well as biophysical transport characteristics obtained from Xenopus oocytes expressing ZmALMT1 indicate that this transporter is implicated in the selective transport of anions involved in mineral nutrition and ion homeostasis processes, rather than mediating a specific Al-activated citrate exudation response at the rhizosphere of maize roots.

The pipeline for macro- and microarray analyses (PMmA) is a set of scripts with a web interface developed to analyze DNA array data generated by array image quantification software. PMmA is designed for use with single- or double-color array data and to work as a pipeline in five classes (data format, normalization, data analysis, clustering, and array maps). It can also be used as a plugin in the BioArray Software Environment, an open-source database for array analysis, or used in a local version of the web service. All scripts in PMmA were developed in the PERL programming language and statistical analysis functions were implemented in the R statistical language. Consequently, our package is a platform-independent software. Our algorithms can correctly select almost 90% of the differentially expressed genes, showing a superior performance compared to other methods of analysis. The pipeline software has been applied to 1536 expressed sequence tags macroarray public data of sugarcane exposed to cold for 3 to 48 h. PMmA identified thirty cold-responsive genes previously unidentified in this public dataset. Fourteen genes were up-regulated, two had a variable expression and the other fourteen were down-regulated in the treatments. These new findings certainly were a consequence of using a superior statistical analysis approach, since the original study did not take into account the dependence of data variability on the average signal intensity of each gene. The web interface, supplementary information, and the package source code are available, free, to non-commercial users at http://ipe.cbmeg.unicamp.br/pub/PMmA.

Sugarcane is a highly productive crop used for centuries as the main source of sugar and recently to produce ethanol, a renewable bio-fuel energy source. There is increased interest in this crop due to the impending need to decrease fossil fuel usage. Sugarcane has a highly polyploid genome. Expressed sequence tag (EST) sequencing has significantly contributed to gene discovery and expression studies used to associate function with sugarcane genes. A significant amount of data exists on regulatory events controlling responses to herbivory, drought, and phosphate deficiency, which cause important constraints on yield and on endophytic bacteria, which are highly beneficial. The means to reduce drought, phosphate deficiency, and herbivory by the sugarcane borer have a negative impact on the environment. Improved tolerance for these constraints is being sought. Sugarcane's ability to accumulate sucrose up to 16% of its culm dry weight is a challenge for genetic manipulation. Genome-based technology such as cDNA microarray data indicates genes associated with sugar content that may be used to develop new varieties improved for sucrose content or for traits that restrict the expansion of the cultivated land. The genes can also be used as molecular markers of agronomic traits in traditional breeding programs.

Cold-set whey protein isolate gels (7% w/w) were produced by the addition of different amounts of glucono-δ-lactone to thermally denatured protein solutions. After 48 h of incubation at 10 ◦ C, different final pH values were obtained (5.2 to 3.9). The gels were analysed by uniaxial com- pression measurements, water-holding capacity and protein solubility. The water-holding capacity of the gels decreased at pH values near the pI, this being associated with the lower protein solubility at this pH value. Protein solubility in the presence or absence of denaturing and reducing agents indicated that electrostatic interactions were responsible for the maintenance of the acidified gel structure at pH values from 5.2 to 4.6, but at pH 4.2, more hydrophobic interactions were present in relation to other final gel pHs, in spite of β-lactoglobulin (β-Lg) and BSA being far from their pI values. Complementary PAGE assays showed that disulphide bonds were associated with inter- nal stabilisation of the protein aggregates formed during heat treatment. The mechanical properties of the gels were influenced by the final system pH value, showing that the strongest network was observed at pH 5.2. The results allowed the conclusion that although the interactions amongst the aggregates in the network of cold-set whey protein gels were essentially the same at the pH values evaluated, β-Lg played an important role in gel stabilisation due to its high concentration in WPI, strengthening the structure at its pI and, at the same time, providing good water-retention capacity. whey proteins / glucono-delta-lactone / cold gelation / protein interactions

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2014-02-26T17:29:02Z No. of bitstreams: 1 WOS000245799300001.pdf: 721026 bytes, checksum: a95bd81c2e9b89892963ba8568987d0e (MD5) Made available in DSpace on 2014-02-26T17:29:02Z (GMT). No. of bitstreams: 1 WOS000245799300001.pdf: 721026 bytes, checksum: a95bd81c2e9b89892963ba8568987d0e (MD5) Previous issue date: 2007-03-13 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:17:30Z No. of bitstreams: 1 WOS000245799300001.pdf: 721026 bytes, checksum: a95bd81c2e9b89892963ba8568987d0e (MD5) Made available in DSpace on 2014-05-20T13:17:30Z (GMT). No. of bitstreams: 1 WOS000245799300001.pdf: 721026 bytes, checksum: a95bd81c2e9b89892963ba8568987d0e (MD5) Previous issue date: 2007-03-13 Background: Sugarcane is an increasingly economically and environmentally important C4 grass, used for the production of sugar and bioethanol, a low-carbon emission fuel. Sugarcane originated from crosses of Saccharum species and is noted for its unique capacity to accumulate high amounts of sucrose in its stems. Environmental stresses limit enormously sugarcane productivity worldwide. To investigate transcriptome changes in response to environmental inputs that alter yield we used cDNA microarrays to profile expression of 1,545 genes in plants submitted to drought, phosphate starvation, herbivory and N-2-fixing endophytic bacteria. We also investigated the response to phytohormones (abscisic acid and methyl jasmonate). The arrayed elements correspond mostly to genes involved in signal transduction, hormone biosynthesis, transcription factors, novel genes and genes corresponding to unknown proteins.Results: Adopting an outliers searching method 179 genes with strikingly different expression levels were identified as differentially expressed in at least one of the treatments analysed. Self Organizing Maps were used to cluster the expression profiles of 695 genes that showed a highly correlated expression pattern among replicates. The expression data for 22 genes was evaluated for 36 experimental data points by quantitative RT-PCR indicating a validation rate of 80.5% using three biological experimental replicates. The SUCAST Database was created that provides public access to the data described in this work, linked to tissue expression profiling and the SUCAST gene category and sequence analysis. The SUCAST database also includes a categorization of the sugarcane kinome based on a phylogenetic grouping that included 182 undefined kinases.Conclusion: An extensive study on the sugarcane transcriptome was performed. Sugarcane genes responsive to phytohormones and to challenges sugarcane commonly deals with in the field were identified. Additionally, the protein kinases were annotated based on a phylogenetic approach. The experimental design and statistical analysis applied proved robust to unravel genes associated with a diverse array of conditions attributing novel functions to previously unknown or undefined genes. The data consolidated in the SUCAST database resource can guide further studies and be useful for the development of improved sugarcane varieties. Univ São Paulo, Inst Quim, Dept Bioquim, BR-01498 São Paulo, Brazil Univ São Paulo, BIOINFO Nucl Pesquisas & Bioinformat, BR-05508 São Paulo, Brazil Univ Estadual Campinas, Ctr Biol Mol & Engn Genet, BR-13081970 Campinas, SP, Brazil Univ Fed Rio de Janeiro, Inst Bioquim Med, BR-21941 Rio de Janeiro, Brazil Univ São Paulo, Escola Super Agr Luiz Queirzo, Dept Genet, BR-05508 Piracicaba, SP, Brazil Ctr Tecnol Canavieira, Piracicaba, SP, Brazil Univ Estadual Paulista, Fac Ciências Agrarias & Vet Jaboticabal, Dept Tecnol, Jaboticabal, SP, Brazil Univ São Paulo, CENA, BR-05508 São Paulo, Brazil Univ Estadual Paulista, Fac Ciências Agrarias & Vet Jaboticabal, Dept Tecnol, Jaboticabal, SP, Brazil

Several advanced techniques have been proposed for data clustering and many of them have been applied to gene expression data, with partial success. The high dimensionality and the multitude of admissible perspectives for data analysis of gene expression require additional computational resources, such as hierarchical structures and dynamic allocation of resources. We present an immune-inspired hierarchical clustering device, called hierarchical artificial immune network (HaiNet), especially devoted to the analysis of gene expression data. This technique was applied to a newly generated data set, involving maize plants exposed to different aluminum concentrations. The performance of the algorithm was compared with that of a self-organizing map, which is commonly adopted to deal with gene expression data sets. More consistent and informative results were obtained with HaiNet.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:21:43Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:44:26Z : No. of bitstreams: 1 2-s2.0-30844463001.pdf: 236130 bytes, checksum: 82f2a0dd0939fb25b9a23f2e2f9ea5a2 (MD5) Made available in DSpace on 2014-05-27T11:21:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) 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. Copyright by the Brazilian Society of Genetics. Universidade Estadual de Campinas Centro de Biologia Molecular e Engenharia Genética, Caixa Postal 6010, 13083-875 Campinas, SP Universidade Estadual de Campinas Instituto de Biologia Departamento de Genética e Evolução, Caixa Postal 6010, 13083-875 Campinas, SP Universidade Estadual Paulista Júlio de Mesquita Filho Instituto de Biociências Departamento de Genética, Botucatu, SP Universidade Estadual Paulista Júlio de Mesquita Filho Instituto de Biociências Departamento de Genética, Botucatu, SP FAPESP: 03/00724-7

Transposable elements (TEs) are considered to be important components of the maintenance and diversification of genomes. The recent increase in genome sequence data has created an opportunity to evaluate the impact of these active mobile elements on the evolution of plant genomes. Analysis of the sugarcane transcriptome identified 267 clones with significant similarity to previously described plant TEs. After full cDNA sequencing, 68 sugarcane TE clones were assigned to 11 families according to their best sequence alignment against a fully characterized element. Expression was further investigated through a combined study utilizing electronic Northerns, macroarray, transient and stable sugarcane transformation. Newly synthesized cDNA probes from flower, leaf roll, apical meristem and callus tissues confirm previous results. Callus was identified as the tissue with the highest number of TEs being expressed, revealing that tissue culture drastically induced the expression of different elements. No tissue-specific family was identified. Different representatives within a TE family displayed differential expression patterns, showing that each family presented expression in almost every tissue. Transformation experiments demonstrated that most Hopscotch clone-derived U3 regions are, indeed, active promoters, although under a strong transcriptional regulation. This is a large-scale study about the expression pattern of TEs and indicates that mobile genetic elements are transcriptionally active in the highly polyploid and complex sugarcane genome.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2014-02-26T17:22:38Z No. of bitstreams: 1 WOS000230919700003.pdf: 708129 bytes, checksum: 81dfe301960a8a0aef14a8a21d2abd09 (MD5) Made available in DSpace on 2014-02-26T17:22:38Z (GMT). No. of bitstreams: 1 WOS000230919700003.pdf: 708129 bytes, checksum: 81dfe301960a8a0aef14a8a21d2abd09 (MD5) Previous issue date: 2005-02-28 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:17:32Z No. of bitstreams: 1 WOS000230919700003.pdf: 708129 bytes, checksum: 81dfe301960a8a0aef14a8a21d2abd09 (MD5) Made available in DSpace on 2014-05-20T13:17:32Z (GMT). No. of bitstreams: 1 WOS000230919700003.pdf: 708129 bytes, checksum: 81dfe301960a8a0aef14a8a21d2abd09 (MD5) Previous issue date: 2005-02-28 A collection of 237,954 sugarcane ESTs was examined in search of signal transduction genes. Over 3,500 components involved in several aspects of signal transduction, transcription, development, cell cycle, stress responses and pathogen interaction were compiled into the Sugarcane Signal Transduction (SUCAST) Catalogue. Sequence comparisons and protein domain analysis revealed 477 receptors, 510 protein kinases, 107 protein phosphatases, 75 small GTPases, 17 G-proteins, 114 calcium and inositol metabolism proteins, and over 600 transcription factors. The elements were distributed into 29 main categories subdivided into 409 sub-categories. Genes with no matches in the public databases and of unknown function were also catalogued. A cDNA microarray was constructed to profile individual variation of plants cultivated in the field and transcript abundance in six plant organs (flowers, roots, leaves, lateral buds, and 1(st) and 4(th) internodes). From 1280 distinct elements analyzed, 217 (17%) presented differential expression in two biological samples of at least one of the tissues tested. A total of 153 genes (12%) presented highly similar expression levels in all tissues. A virtual profile matrix was constructed and the expression profiles were validated by real-time PCR. The expression data presented can aid in assigning function for the sugarcane genes and be useful for promoter characterization of this and other economically important grasses. Univ São Paulo, Inst Quim, Dept Bioquim, BR-05508900 São Paulo, Brazil Univ São Paulo, Inst Matemat & Estatist, Dept Estatist, BR-05508090 São Paulo, Brazil Univ Estadual Campinas, Inst Biol, Dept Genet & Evolucao, BR-13083970 Campinas, SP, Brazil Univ Estadual Campinas, Ctr Biol Mol & Engn Genet, BR-13083970 Campinas, SP, Brazil Ctr Tecnol Copersucar, BR-13400970 São Paulo, Brazil Univ Estadual Paulista, Fac Ciências Agr & Vet Jaboticabal, Dept Tecnol, Ctr Estocagem Clones,BCCCtr, BR-14870000 São Paulo, Brazil Univ Estadual Paulista, Fac Ciências Agr & Vet Jaboticabal, Dept Tecnol, Ctr Estocagem Clones,BCCCtr, BR-14870000 São Paulo, Brazil