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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.

Plants are sessile organisms and have evolved to tolerate a constantly changing environment. After the onset of different stress conditions, calcineurin B-like (CBL) proteins can sense calcium signals and activate CBL-interacting protein kinase (CIPK) proteins, which can phosphorylate downstream proteins to reestablish plant homeostasis. Previous studies in the bioenergy crop sugarcane showed that the ScCIPK8 gene is induced by drought stress and is also related to sucrose content. Here, we have characterized the protein-protein interactions of ScCIPK8 with six CBL proteins (ScCBL1, ScCBL2, ScCBL3, ScCBL6, ScCBL9, and ScCBL10). Yeast two-hybrid assays showed that ScCIPK8 interacts with ScCBL1, ScCBL3, and ScCBL6. Bimolecular fluorescence complementation assays confirmed in planta the interactions that were observed in yeast cells. These findings give insights on the regulatory networks related to sugar accumulation and drought stress responses in sugarcane.

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.

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.

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.

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.

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.

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.

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

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.

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.

BackgroundDrought is a major abiotic stress that affects crop productivity worldwide. Sugarcane can withstand periods of water scarcity during the final stage of culm maturation, during which sucrose accumulation occurs. Meanwhile, prolonged periods of drought can cause severe plant losses.Methodology/principal findingsIn a previous study, we evaluated the transcriptome of drought-stressed plants to better understand sugarcane responses to drought. Among the up-regulated genes was Scdr1 (sugarcane drought-responsive 1). The aim of the research reported here was to characterize this gene. Scdr1 encodes a putative protein containing 248 amino acids with a large number of proline (19%) and cysteine (13%) residues. Phylogenetic analysis showed that ScDR1is in a clade with homologs from other monocotyledonous plants, separate from those of dicotyledonous plants. The expression of Scdr1 in different varieties of sugarcane plants has not shown a clear association with drought tolerance.Conclusions/significanceThe overexpression of Scdr1 in transgenic tobacco plants increased their tolerance to drought, salinity and oxidative stress, as demonstrated by increased photosynthesis, water content, biomass, germination rate, chlorophyll content and reduced accumulation of ROS. Physiological parameters, such as transpiration rate (E), net photosynthesis (A), stomatal conductance (gs) and internal leaf CO(2) concentration, were less affected by abiotic stresses in transgenic Scdr1 plants compared with wild-type plants. Overall, our results indicated that Scdr1 conferred tolerance to multiple abiotic stresses, highlighting the potential of this gene for biotechnological applications.

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.

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.

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.

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

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

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

Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America. Pathogenicity appears to be intimately related to the dimorphic transition from the hyphal to the yeast form, which is induced by a shift from environmental temperature to the temperature of the mammalian host. Little information is available on the P. brasiliensis genes that are necessary during the pathogenic phase. We have therefore undertaken Suppression Subtraction Hybridization (SSH) and macroarray analyses with the aim of identifying genes that are preferentially expressed in the yeast phase. Genes identified by both procedures as being more highly expressed in the yeast phase are involved in basic metabolism, signal transduction, growth and morphogenesis, and sulfur metabolism. In order to test whether the observed changes in gene expression reflect the differences between the growth conditions used to obtain the two morphological forms rather than differences intrinsic to the cell types, we performed real-time RT-PCR experiments using RNAs derived from both yeast cells and mycelia that had been cultured at 37 degrees C and 26 degrees C in either complete medium (YPD or Sabouraud) or minimal medium. Twenty genes, including AGS1 (alpha-1,3-glucan synthase) and TSA1 (thiol-specific antioxidant), were shown to be more highly expressed in the yeast cells than in the hyphae. Although their levels of expression could be different in rich and minimal media, there was a general tendency for these genes to be more highly expressed in the yeast cells.

Two cDNA clones homologous to myrosinase-binding proteins (MBPs) were identified by differential display in Arabidopsis thaliana (L.) Heynh. The cDNAs (MBP1 and MBP2) correspond to two open-reading frames found in a gene cluster of seven putative MBP genes located on chromosome 1. The predicted proteins MBP1 and MBP2 are similar to lectins and plant aggregating factors. In addition, MBP2 contains a region of high content of proline and alanine residues, commonly found in arabinogalactan proteins and hydroxyproline-rich glycoproteins. Transcripts corresponding to MBP1 and MBP2 genes are exclusively and abundantly expressed in flowers but are not detected in male-sterile flowers of coi1 plants, insensitive to jasmonic acid. Northern analysis and in situ hybridization revealed that MBP mRNAs are present in higher levels in immature flowers and are localized in several floral organs, including the ovary, ovules, style, anthers and filament. Transcripts of the Arabidopsis myrosinase gene TGG1 show a pattern of expression similar to that observed for the MBP genes during flower development; however, they are also abundant in green tissues and are only partially affected by COI1. Crude preparations of soluble proteins from leaf and flower extracts of wild-type Arabidopsis showed myrosinase activity when sinigrin was used as substrate. In contrast, coi1 plants showed significantly reduced myrosinase activities in both leaves and flowers. The results show that COI1 controls MBP expression in flowers and significantly affects the expression and activity of myrosinase in Arabidopsis.

The cloning and sequence analysis of a novel gene that encodes a type 2 non-specific lipid transfer-like protein (LTP) from rice is reported. Sequence analysis revealed an ORF encoding a protein showing characteristics of the LTP proteins. However, rice LTP2 is more similar to heterologous LTPs than to rice LTP1, supporting the existence of two distinct families of plant LTPs. Ltp2 mRNA is accumulated only in mature seeds. In vegetative tissues, mRNA was only detected after treatment with abscisic acid (ABA), mannitol or NaCl. Transient expression experiments that the 61 nucleotides upstream of the TATA box, containing two ACGT boxes and the motif I, are sufficient for ABA responsiveness of the Ltp gene., This work was supported by a post-doctoral fellowship from the Consejo Superior de Investigaciones Cientificas (C.S.I.C.) of Spain to J.M. Garcı́a-Garrido, the Centre Français du Riz (Arles) to M.D. and Dirección General de Polı́tica Cientı́fica of Spanish MEC (PB93-0043) to J.A.M.I. M.M. is a fellow of CNPq (Brazil). The work has been carried out within the framework of Laboratorio Europeo Asociado (LEA) between the laboratories of Perpignan (CNRS) and Barcelona (CSIC).

Sugarcane (Saccharum spp.) is one of the most important crops in the world. Drought stress is a major abiotic stress factor that significantly reduces sugarcane yields. However the gene network that mediates plant responses to water stress remains largely unknown in several crop species. Although several microRNAs that mediate post-transcriptional regulation during water stress have been described in other species, the role of the sugarcane microRNAs during drought stress has not been studied. The objective of this work was to identify sugarcane miRNAs that are differentially expressed under drought stress and to correlate this expression with the behavior of two sugarcane cultivars with different drought tolerances. The sugarcane cultivars RB867515 (higher drought tolerance) and RB855536 (lower drought tolerance) were cultivated in a greenhouse for three months and then subjected to drought for 2, 4, 6 or 8 days. By deep sequencing of small RNAs, we were able to identify 18 miRNA families. Among all of the miRNAs thus identified, seven were differentially expressed during drought. Six of these miRNAs were differentially expressed at two days of stress, and five miRNAs were differentially expressed at four days. The expression levels of five miRNAs (ssp-miR164, ssp-miR394, ssp-miR397, ssp-miR399-seq 1 and miR528) were validated by RT-qPCR (quantitative reverse transcriptase PCR). Six precursors and the targets of the differentially expressed miRNA were predicted using an in silico approach and validated by RT-qPCR; many of these targets may play important roles in drought tolerance. These findings constitute a significant increase in the number of identified miRNAs in sugarcane and contribute to the elucidation of the complex regulatory network that is activated by drought stress.

Background The nucleotide sequence flanking the translation initiation codon (start codon context) affects the translational efficiency of eukaryotic mRNAs, and may indicate the presence of an alternative translation initiation site (TIS) to produce proteins with different properties. Multi-targeting may reflect the translational variability of these other protein forms. In this paper we present a web server that performs computations to investigate the usage of alternative translation initiation sites for the synthesis of new protein variants that might have different functions. Results An efficient web-based tool entitled TISs-ST (Translation Initiation Sites and Secretory Targets) evaluates putative translation initiation sites and indicates the prediction of a signal peptide of the protein encoded from this site. The TISs-ST web server is freely available to both academic and commercial users and can be accessed at http://ipe.cbmeg.unicamp.br/pub/TISs-ST. Conclusion The program can be used to evaluate alternative translation initiation site consensus with user-specified sequences, based on their composition or on many position weight matrix models. TISs-ST provides analytical and visualization tools for evaluating the periodic frequency, the consensus pattern and the total information content of a sequence data set. A search option allows for the identification of signal peptides from predicted proteins using the PrediSi software.

Sugarcane (Saccharum spp.) is the most promising crop for renewable energy. Among the diverse stresses that affect plant productivity, drought stress frequently causes losses in sugarcane fields. Although several studies have addressed plant responses to drought using controlled environments, plant responses under field conditions are largely unknown. Recently, microRNA (miRNA)-mediated post-transcriptional regulation has been described as an important and decisive component in vegetal development and stress resistance modulation. The role of miRNAs in sugarcane responses to drought under field conditions is currently not known. Two sugarcane cultivars differing in drought tolerance were grown in the field with and without irrigation (rainfed) for 7 months. By using small RNA deep sequencing, we were able to identify 18 miRNA families comprising 30 mature miRNA sequences. Among these families, we found 13 mature miRNAs that were differentially expressed in drought-stressed plants. Seven miRNAs were differentially expressed in both cultivars. The target genes for many of the differentially expressed mature miRNAs were predicted, and some of them were validated by quantitative reverse transcription PCR. Among the targets, we found transcription factors, transporters, proteins associated with senescence, and proteins involved with flower development. All of these data increase our understanding of the role of miRNAs in the complex regulation of drought stress in field-grown sugarcane, providing valuable tools to develop new sugarcane cultivars tolerant to drought stress. Electronic supplementary material The online version of this article (doi:10.1007/s00425-012-1795-7) contains supplementary material, which is available to authorized users.

Background - Sucrose content is a highly desirable trait in sugarcane as the worldwide demand for cost-effective biofuels surges. Sugarcane cultivars differ in their capacity to accumulate sucrose and breeding programs routinely perform crosses to identify genotypes able to produce more sucrose. Sucrose content in the mature internodes reach around 20% of the culms dry weight. Genotypes in the populations reflect their genetic program and may display contrasting growth, development, and physiology, all of which affect carbohydrate metabolism. Few studies have profiled gene expression related to sugarcane's sugar content. The identification of signal transduction components and transcription factors that might regulate sugar accumulation is highly desirable if we are to improve this characteristic of sugarcane plants. Results - We have evaluated thirty genotypes that have different Brix (sugar) levels and identified genes differentially expressed in internodes using cDNA microarrays. These genes were compared to existing gene expression data for sugarcane plants subjected to diverse stress and hormone treatments. The comparisons revealed a strong overlap between the drought and sucrose-content datasets and a limited overlap with ABA signaling. Genes associated with sucrose content were extensively validated by qRT-PCR, which highlighted several protein kinases and transcription factors that are likely to be regulators of sucrose accumulation. The data also indicate that aquaporins, as well as lignin biosynthesis and cell wall metabolism genes, are strongly related to sucrose accumulation. Moreover, sucrose-associated genes were shown to be directly responsive to short term sucrose stimuli, confirming their role in sugar-related pathways. Conclusion - Gene expression analysis of sugarcane populations contrasting for sucrose content indicated a possible overlap with drought and cell wall metabolism processes and suggested signaling and transcriptional regulators to be used as molecular markers in breeding programs. Transgenic research is necessary to further clarify the role of the genes and define targets useful for sugarcane improvement programs based on transgenic plants.