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Background: Sugarcane (Saccharum spp.) covers vast areas of land (around 25 million ha worldwide), and its processing is already linked into infrastructure for producing bioethanol in many countries. This makes it an ideal candidate for improving composition of its residues (mostly cell walls), making them more suitable for cellulosic ethanol production. In this paper, we report an approach to improving saccharification of sugarcane straw by RNAi silencing of the recently discovered BAHD01 gene responsible for feruloylation of grass cell walls. Results: We identified six BAHD genes in the sugarcane genome (SacBAHDs) and generated five lines with substantially decreased SacBAHD01 expression. To find optimal conditions for determining saccharification of sugarcane straw, we tried multiple combinations of solvent and temperature pretreatment conditions, devising a predictive model for finding their effects on glucose release. Under optimal conditions, demonstrated by Organosolv pretreatment using 30% ethanol for 240 min, transgenic lines showed increases in saccharification efficiency of up to 24%. The three lines with improved saccharification efficiency had lower cell-wall ferulate content but unchanged monosaccharide and lignin compositions. Conclusions: The silencing of SacBAHD01 gene and subsequent decrease of cell-wall ferulate contents indicate a promising novel biotechnological approach for improving the suitability of sugarcane residues for cellulosic ethanol production. In addition, the Organosolv pretreatment of the genetically modified biomass and the optimal conditions for the enzymatic hydrolysis presented here might be incorporated in the sugarcane industry for bioethanol production. Keywords: Sugarcane, Made available in DSpace on 2019-06-22T00:35:25Z (GMT). No. of bitstreams: 1 SilencingofaBAHDacyltransferase.pdf: 1991226 bytes, checksum: 2c98f01c74be1c72d0b268e90455a2cb (MD5) Previous issue date: 2019

BackgroundA major limiting factor for plant growth is the aluminum (Al) toxicity in acidic soils, especially in tropical regions. The exclusion of Al from the root apex through root exudation of organic acids such as malate and citrate are both the most ubiquitous tolerance mechanisms in the plant kingdom. Two families of anion channels that confer Al tolerance are well described in the literature, ALMT and MATE family. ResultsIn this study, sugarcane plants constitutively overexpressing the Sorghum bicolor MATE gene (SbMATE) showed improved tolerance to Al when compared to non-transgenic (NT) plants, characterized by sustained root growth and exclusion of aluminum from the root apex based on the result obtained with hematoxylin staining. In addition, genome-wide analysis of the recently released sugarcane genome identified 11 ALMT genes and molecular studies showed potential new targets for aluminum tolerance. ConclusionsOur results indicate that the transgenic plants overexpressing the Sorghum bicolor MATE has an improved tolerance to Al. The expression profile of ALMT genes revels potential candidate genes to be used has an alternative for agricultural expansion in Brazil and other areas with aluminum toxicity in poor and acid soils.

Grass cell walls have hydroxycinnamic acids attached to arabinosyl residues of arabinoxylan (AX), and certain BAHD acyltransferases are involved in their addition. In this study, we characterized one of these BAHD genes in the cell wall of the model grass Setaria viridis. RNAi silenced lines of S. viridis (SvBAHD05) presented a decrease of up to 42% of ester-linked p-coumarate (pCA) and 50% of pCA-arabinofuranosyl, across three generations. Biomass from SvBAHD05 silenced plants exhibited up to 32% increase in biomass saccharification after acid pre-treatment, with no change in total lignin. Molecular dynamics simulations suggested that SvBAHD05 is a p-coumaroyl coenzyme A transferase (PAT) mainly involved in the addition of pCA to the arabinofuranosyl residues of AX in Setaria. Thus, our results provide evidence of p-coumaroylation of AX promoted by SvBAHD05 acyltransferase in the cell wall of the model grass S. viridis. Furthermore, SvBAHD05 is a promising biotechnological target to engineer crops for improved biomass digestibility for biofuels, biorefineries and animal feeding.

International audience; Climate change is posing a major challenge to coffee production worldwide leading to a need for the development of coffee cultivars with increased drought tolerance. In several plant species, the use of DREB genes in crop improvement has achieved promising results to desiccation tolerance engineering. Recent studies reported CcDREB1D specific patterns of expression in Coffea canephora and functional evidence of this gene involvement in drought stress responses. However, knowledge on natural diversity of this gene is largely unknown. In this context, this study aimed at evaluating the sequence variability of the DREB1D gene in several Coffea genotypes. Nucleotide variation in promoters and coding regions of this gene were evaluated in a population consisting of 38 genotypes of C. canephora, C. arabica and C. eugenioides, most of them characterized by different phenotypes (tolerance vs. susceptibility) in relation to drought. The genetic diversity of the loci revealed different haplotypes for the promoter and coding regions. In particular, our findings suggest association between drought tolerance and the genetic variations on DREB1D promoter regions, but not with those from its corresponding coding regions. Gene expression studies revealed up-regulated expression of DREB1D gene upon drought mainly in leaves of drought-tolerant clones of C. canephora, and in response to drought, high, and low temperatures in leaves of C. arabica, suggesting a key role of this gene in coffee responses to abiotic stress.

Sugarcane (Saccharum spp.) is a monocotyledonous semi-perennial C4 grass of the Poaceae family. Its capacity to accumulate high content of sucrose and biomass makes it one of the most important crops for sugar and biofuel production. Conventional methods of sugarcane breeding have shown several limitations due to its complex polyploid and aneuploid genome. However, improvement by biotechnological engineering is currently the most promising alternative to introduce economically important traits. In this work, we present an improved protocol for Agrobacterium tumefaciens-mediated transformation of commercial sugarcane hybrids using immature top stalk-derived embryogenic callus cultures. The callus cultures are transformed with preconditioned A. tumefaciens carrying a binary vector that encodes expression cassettes for a gene of interest and the bialaphos resistance gene (bar confers resistance to glufosinate-ammonium herbicide). This protocol has been used to successfully transform a commercial sugarcane cultivar, SP80-3280, highlighting: (i) reduced recalcitrance and oxidation; (ii) high yield of embryogenic callus; (iii) improved selection; and (iv) shoot regeneration and rooting of the transformed plants. Altogether, these improvements generated a transformation efficiency of 2.2%. This protocol provides a reliable tool for a routine procedure for sugarcane improvement by genetic engineering. © 2017 by John Wiley & Sons, Inc.

Feruloylation of arabinoxylan (AX) in grass cell walls is a key determinant of recalcitrance to enzyme attack, making it a target for improvement of grass crops, and of interest in grass evolution. Definitive evidence on the genes responsible is lacking so we studied a candidate gene that we identified within the BAHD acyl-CoA transferase family. We used RNA interference (RNAi) silencing of orthologs in the model grasses Setaria viridis (SvBAHD01) and Brachypodium distachyon (BdBAHD01) and determined effects on AX feruloylation. Silencing of SvBAHD01 in Setaria resulted in a c. 60% decrease in AX feruloylation in stems consistently across four generations. Silencing of BdBAHD01 in Brachypodium stems decreased feruloylation much less, possibly due to higher expression of functionally redundant genes. Setaria SvBAHD01 RNAi plants showed: no decrease in total lignin, approximately doubled arabinose acylated by p-coumarate, changes in two-dimensional NMR spectra of unfractionated cell walls consistent with biochemical estimates, no effect on total biomass production and an increase in biomass saccharification efficiency of 40-60%. We provide the first strong evidence for a key role of the BAHD01 gene in AX feruloylation and demonstrate that it is a promising target for improvement of grass crops for biofuel, biorefining and animal nutrition applications.

SUMMARYControlled irrigation during the dry period associated with adequate nitrogen (N), phosphorous (P) and potassium (K) fertilization led to the accumulation of biochemical compounds in coffee beans considered as positive precursors of beverage quality. Adult plants of coffee (Coffea arabica ‘Rubi’) were cultivated using different water regimes (WR) and fertilization conditions under the dry climate of the Brazilian Cerrado. Coffee-bean physical characteristics were evaluated as well as biochemical composition by near-infrared analysis. The K treatment mostly affected bean biochemistry, lipid and chlorogenic acid (CGA) contents, which increased with increasing amounts of fertilizer. Caffeine contents increased with higher amounts of N, but no significant effects of P treatment on bean biochemical composition were observed. Sucrose and total lipid contents always appeared higher in beans of non-irrigated plants than those beans from plants grown with continuous irrigation. In contrast, caffeine and CGA contents were higher in beans of irrigated as compared with non-irrigated plants. For the first time, the current results showed that controlled management of irrigation during the dry period associated with reasonable NPK fertilization led to the accumulation of biochemical compounds in coffee beans considered as positive precursors of beverage quality.

After the publication of the article [1], it was brought to our attention that the GenBank Accession number for the EF1 is missing in Methods section. Please find the accession number in the erratum below.

The effects of water deficit on photochemical parameters and expression of several candidate genes were investigated in drought-tolerant clone 73 of Coffea canephora submitted to slowly imposed water limitation. Under irriga- tion, this clone showed low values of stomatal conductance (gs) and of CO2 assimilation rates (A )s uggesting that it had a great efficiency in controlling stomatal closure and transpira- tion. After water withdrawal, this clone reached a −3.0 MPa after 15days without irrigation and showeda slow decrease in the pre-dawn leaf water potential. Under drought, the suppres- sion of Awas accompanied by maintenance of photochemical quenching (qP) and internal to ambient CO2 concentration (Ci/Ca) ratios as well as by a decrease of non-photochemical quenching (qN). This is confirmed by the transport rate/CO2 assimilation (ETR/A) rates that suggested the participation of an alternative electron sink protecting the photosynthetic apparatus against photoinhibition. At the transcriptomic level, high up-regulation of genes encoding for a dehydrin (CcDH3), an ascorbate peroxidase (CcAPX1), a prephenate-dehydrogenase like protein (CcPDH1) and a non-symbiotic haemoglobin (CcNSH1) was also observed upon drought suggesting a strong induction of antioxidant and osmoprotection systems in this clone. High expression levels of gene-encoding ABA receptors (CcPYL3 and CcPYL7) under water limitation were also ob- served suggesting the involvement of the ABA signaling path- way in response to drought. All these results where compared to those previously obtained for drought-tolerant clones 14 and 120. Our results demonstrated the existence of different mecha- nisms amongst the drought-tolerant coffee clones regarding water deficit.

Most of the world's coffee production originates from Coffea arabica, an allotetraploid species with low genetic diversity and for which few genomic resources are available. Genomic libraries with large DNA fragment inserts are useful tools for the study of plant genomes, including the production of physical maps, integration studies of physical and genetic maps, genome structure analysis and gene isolation by positional cloning. Here, we report the construction and characterization of a Bacterial Artificial Chromosome (BAC) library from C. arabica Timor Hybrid CIFC 832/2, a parental genotype for several modern coffee cultivars. The BAC library consists of 56,832 clones with an average insert size of 118 kb, which represents a dihaploid genome coverage of five to sixfold. The content of organellar DNA was estimated at 1.04 and 0.5 % for chloroplast and mitochondrial DNA, respectively. The BAC library was screened for the NADPH-dependent mannose-6-phosphate reductase gene (CaM6PR) with markers positioned on four linkage groups of a partial C. arabica genetic map. A mixed approach using PCR and membrane hybridization of BAC pools allowed for the discovery of nine BAC clones with the CaM6PR gene and 53 BAC clones that were anchored to the genetic map with simple sequence repeat markers. This library will be a useful tool for future studies on comparative genomics and the identification of genes and regulatory elements controlling major traits in this economically important crop species.

Background Second-generation ethanol (2G-bioethanol) uses lignocellulosic feedstocks for ethanol production. Sugarcane is one among the most suitable crops for biofuel production. Its juice is extracted for sugar production, while sugarcane bagasse, straw, and senescing leaves are considered industrial waste. Senescence is the age-dependent deterioration of plant cells, ultimately leading to cell death and completion of the plant life cycle. Because senescing leaves may also be used for biofuel production, understanding the process of natural senescence, including remobilization of nutrients and its effect on cell walls can provide useful information for 2G-bioethanol production from sugarcane leaves. Results The natural senescence process in leaves of the commercial sugarcane cultivar RB867515 was investigated. Senescence was characterized by strong reduction in photosynthetic pigments content, remobilization of the nutrients N, P, K, B, Cu, Fe, and Zn, and accumulation of Ca, S, Mg, B, Mn, and Al. No significant changes in the cell-wall composition occurred, and only small changes in the expression of cell wall-related genes were observed, suggesting that cell walls are preserved during senescence. Senescence-marker genes, such as SAG12-like and XET-like genes, were also identified in sugarcane and found to be highly expressed. Conclusions Our study on nutrient remobilization under senescence in a vigorous sugarcane cultivar can contribute to the understanding on how nutrient balance in a high-yielding crop is achieved. In general, neutral monosaccharide profile did not change significantly with leaf senescence, suggesting that senescing leaves of sugarcane can be as a feedstock for biofuel production using pretreatments established for non-senescing leaves without additional efforts. Based on our findings, the potential biotechnological applications for the improvement of sugarcane cultivars are discussed. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0568-0) contains supplementary material, which is available to authorized users.

Real-time PCR (RT-qPCR) expression analysis is a powerful analytical technique, but reliable results depend on the use of stable reference genes for proper normalization. This study proposed to test the expression stability of 13 candidate reference genes in Setaria viridis, a monocot species recently proposed as a new C4 model plant. Gene expression stability of these genes was assayed across different tissues and developmental stages of Setaria and under drought or aluminum stress. In general, our results showed Protein Kinase, RNA Binding Protein and SDH as the most stable genes. Moreover, pairwise analysis showed that two reference genes were sufficient to normalize the gene expression data under each condition. By contrast, GAPDH and ACT were the least stably expressed genes tested. Validation of suitable reference genes was carried out to profile the expression of P5CS and GolS during abiotic stress. In addition, normalization of gene expression of SuSy, involved in sugar metabolism, was assayed in the developmental dataset. This study provides a list of reliable reference genes for transcript normalization in S. viridis in different tissues and stages of development and under abiotic stresses, which will facilitate genetic studies in this monocot model plant.

The aim of this study was to investigate the molecular mechanisms underlying drought acclimation in coffee plants by the identification of candidate genes (CGs) using different approaches. The first approach used the data generated during the Brazilian Coffee expressed sequence tag (EST) project to select 13 CGs by an in silico analysis (electronic northern). The second approach was based on screening macroarrays spotted with plasmid DNA (coffee ESTs) with separate hybridizations using leaf cDNA probes from drought-tolerant and susceptible clones of Coffea canephora var. Conilon, grown under different water regimes. This allowed the isolation of seven additional CGs. The third approach used two-dimensional gel electrophoresis to identify proteins displaying differential accumulation in leaves of drought-tolerant and susceptible clones of C. canephora. Six of them were characterized by MALDI-TOF-MS/MS (matrix-assisted laser desorption-time of flight-tandem mass spectrometry) and the corresponding proteins were identified. Finally, additional CGs were selected from the literature, and quantitative real-time polymerase chain reaction (qPCR) was performed to analyse the expression of all identified CGs. Altogether, >40 genes presenting differential gene expression during drought acclimation were identified, some of them showing different expression profiles between drought-tolerant and susceptible clones. Based on the obtained results, it can be concluded that factors involved a complex network of responses probably involving the abscisic signalling pathway and nitric oxide are major molecular determinants that might explain the better efficiency in controlling stomata closure and transpiration displayed by drought-tolerant clones of C. canephora.

A novel family of antimicrobial peptides, named raniseptins, has been characterized from the skin secretion of the anuran Hypsiboas raniceps. Nine cDNA molecules have been successfully cloned, sequenced, and their respective polypeptides were characterized by mass spectrometry and Edman degradation. The encoded precursors share structural similarities with the dermaseptin prepropeptides from the Phyllomedusinae subfamily and the mature 28-29 residue long peptides undergo further proteolytic cleavage in the crude secretion yielding consistent fragments of 14-15 residues. The biological assays performed demonstrated that the Rsp-1 peptide has antimicrobial activity against different bacterial strains without significant lytic effect against human erythrocytes, whereas the peptide fragments generated by endoproteolysis show limited antibiotic potency. MALDI imaging mass spectrometry in situ studies have demonstrated that the mature raniseptin peptides are in fact secreted as intact molecules within a defined glandular domain of the dorsal skin, challenging the physiological role of the observed raniseptin fragments, identified only as part of the crude secretion. In this sense, stored and secreted antimicrobial peptides may confer distinct protective roles to the frog.

Tese (doutorado)—Universidade de Brasília, Instituto de Biologia, Departamento de Biologia Celular, Programa de Pós-Graduação em Biologia Molecular, 2015. No que diz respeito à cafeicultura e ao processamento dos grãos de café, o conceito de qualidade do produto foi definido sob diferentes aspectos, como, por exemplo, qualidade de produção, níveis de torra, aroma. Acredita-se que muitos dos constituintes do grão influenciam diretamente nas características organolépticas apresentadas pela bebida. Entretanto, pouco se conhece a respeito de quais moléculas estariam desempenhando tais funções, principalmente, em relação a proteínas e peptídeos encontrados no endosperma da semente do café, uma vez que a maioria dos estudos relacionados focam moléculas não protéicas. Alguns peptídeos que são biologicamente ativos, ou seja, bioativos, estão encriptados em proteínas de origem animal e vegetal e podem ser encontrados com a realização de um screening de um banco de dados utilizando parâmetros pré-definidos. Uma das formas de obtenção de peptídeos bioativos é por meio da ingestão de alimentos que contenham proteínas precursoras e que sofram proteólise por enzimas digestivas. No presente trabalho, foi realizada uma avaliação da composição química de Coffea arabica que visou identificar os precursores de qualidade da bebida do café que estivessem associados a proteínas com possíveis sequências de peptídeos opioides como parte de suas estruturas primárias. Para isso foram realizadas análises in silico em bancos de dados internacionais e na base de dados do Genoma Café, buscando peptídeos que apresentassem a região N-terminal (YGG) similar a outros peptídeos opioides. Outra metodologia utilizada foi a digestão do extrato proteico de Coffea arabica var. Acauã com enzimas digestivas, seguido de fracionamento por HPLC e sequenciamento por Espectrometria de Massa. Os peptídeos encontrados estavam encriptados em uma família de proteínas chamada dehidrina. Esses peptídeos, identificados em ambos métodos, foram sintetizados utilizando-se a estratégia Fmoc/t-butila (9-fluorenilmetoxicarbonila) de síntese manual em suporte sólido e tiveram a atividade (opioide) avaliada em modelos in vivo que permitiram determinar suas funções. Os testes utilizados para avaliar a atividade opioide foram o da placa quente e o da retirada da cauda. Foi possível concluir que as metodologias utilizadas para desvendar os peptídeos encriptados mostrou-se ser eficiente, já que todos os peptídeos testados apresentaram atividade antinociceptiva em camundongos. Regarding to coffee and processing of coffee beans, the product quality concept has been defined in many different ways, for example, production quality, roasting levels, flavor. It is believed that many of the grain constituents directly influence the organoleptic characteristics of the beverage. However, little is known about the molecules performing such functions, especially concerning peptides and proteins found in the endosperm of the coffee seed, since most studies related to this topic focuses on non-protein molecules. Some peptides which are biologically active, ie bioactive, are encrypted in animal or vegetal proteins and can be found by a database screening, using default parameters. One way to obtain bioactive peptides is through the ingestion of foods containing precursor proteins susceptible to proteolysis by digestive enzymes. In the present study, an evaluation of the chemical composition of Coffea arabica was conducted which aimed identifying the coffee beverage quality precursors associated with proteins containing putative sequences of opioid peptides as part of their primary structures. As part of the investigation, in silico analyzes were performed using the international databases and the Coffee Genome database by searching peptides with N-terminal region (YGG) similar to other opioid peptides. Another methodology used was the digestion of protein extract of Coffea arabica var. Acauã with digestive enzymes, followed by HPLC purification and sequencing by Mass Spectrometry. The detected peptides were encrypted in a family of proteins called dehydrin. Those peptides identified in both methods were synthesized by using Fmoc/t-Butyl strategy (9-fluorenilmetoxicarbonila) of manual synthesis on solid support, and their activity (opioid) was evaluated by in vivo models which allowed to determine their roles. The tests used to access the opioid activity were the hot plate and tail flick. It was concluded that the methodologies used to unlock the encrypted peptides have proved to be efficient since all of the tested peptides showed antinociceptive activity in mice.

Six new antimicrobial peptides structurally related to the dermaseptin family have been isolated from the skin secretion of the amphibian Phyllomedusa hypochondrialis. The primary structures of these molecules named as DShypo 01, 02, 03, 04, 06, and 07 were determined by de novo MS/MS experiments, Edman degradation, and cDNA sequencing. The fifth peptide was found to be precisely the same DS 01 from Phyllomedusa oreades previously described by our group. The majority of the peptides purified from the crude skin secretion could be directly localized and mapped onto a freshly dissected dorsal skin fragment using mass spectrometry-imaging techniques. Comparisons between peptides and commercial drugs on their antibacterial and anti-Leishmania amazonensis efficiencies, associated with peptide lytic effects on mammalian blood cells and surface plasmon resonance interaction studies on immobilized DMPC vesicles, were also performed.

Background Bioactive peptides prospection is important for biotechnology field as well as a starting point in many research areas, such as: new drugs development and production of genetically modified plants [1]. In general, bioactive peptides have been identified as candidates for the new drugs development because their intrinsic properties concerning some potential activities, like high specificity, potency, less toxicity, and also chemistry and biological diversity [2]. Peptides can present diverse activities such as antimicrobial, opioids, hypotensive, antithrombotic among others. In 2010, around 60 synthetic peptides with therapeutic potential have been available to pharmaceutical marketing and can be used in many pathologies like, allergies, asthma, arthritis, cardiovascular diseases, diabetes, gastrointestinal dysfunction, growth problems, inflammation, obesity, infectious diseases, cancer, osteoporosis, pain, vaccines and others. [3,4]. For pharmaceutical industry, in therapeutic area, the most promising projects are those that focus on cancer, pain, diabetes, Alzheimer’s disease and depression [5]. This study aims to design sequences of synthetic peptides that may have dual activity: antinociceptive and hypotensive. These peptides act on different targets, opioids receptors and angiotensin converting enzyme (ACE), respectively.

The aim of the present study was to perform a genomic analysis of non-specific lipid-transfer proteins (nsLTPs) in coffee. Several nsLTPs-encoding cDNA and gene sequences were cloned from Coffea arabica and Coffea canephora species. In this work, their analyses revealed that coffee nsLTPs belong to Type II LTP characterized under their mature forms by a molecular weight of around 7.3 kDa, a basic isoelectric points of 8.5 and the presence of typical CXC pattern, with X being an hydrophobic residue facing towards the hydrophobic cavity. Even if several single nucleotide polymorphisms were identified in these nsLTP-coding sequences, 3D predictions showed that they do not have a significant impact on protein functions. Northern blot and RT-qPCR experiments revealed specific expression of Type II nsLTPs-encoding genes in coffee fruits, mainly during the early development of endosperm of both C. arabica and C. canephora. As part of our search for tissue-specific promoters in coffee, an nsLTP promoter region of around 1.2 kb was isolated. It contained several DNA repeats including boxes identified as essential for grain specific expression in other plants. The whole fragment, and a series of 5' deletions, were fused to the reporter gene beta-glucuronidase (uidA) and analyzed in transgenic Nicotiana tabacum plants. Histochemical and fluorimetric GUS assays showed that the shorter (345 bp) and medium (827 bp) fragments of nsLTP promoter function as grain-specific promoters in transgenic tobacco plants.

Spiders produce up to six different kinds of silk, each one for a specific biological function. Spider silks are also known for their unique mechanical properties. The possibility of producing new materials with similar properties motivated research on these silk proteins (spidroins). Using expression sequence tags, we identified four spidroins produced by major ampullate, minor ampullate, flagelliform and tubuliform silk glands from the Brazilian spider Nephilengys cruentata (Araneae: Nephilidae). The new protein sequences showed substantial similarity to other spidroins previously described, with high content of alanine and glycine due to the presence of the highly repetitive motifs (polyAla, (GA)n, (GGX)n, (GPGGX)n). Similarities among sequences were also observed between the different spidroins with the exception of tubuliform spidroin, which presents a unique complex amino acid sequence with high amounts of serine and low amounts of glycine.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T18:43:23Z No. of bitstreams: 1 S1677-04202006000100008.pdf: 927918 bytes, checksum: 9258fae108c1ae62bed73557138b4add (MD5) Made available in DSpace on 2013-08-22T18:43:24Z (GMT). No. of bitstreams: 1 S1677-04202006000100008.pdf: 927918 bytes, checksum: 9258fae108c1ae62bed73557138b4add (MD5) Previous issue date: 2006-03-01 Made available in DSpace on 2013-09-30T17:50:19Z (GMT). No. of bitstreams: 2 S1677-04202006000100008.pdf: 927918 bytes, checksum: 9258fae108c1ae62bed73557138b4add (MD5) S1677-04202006000100008.pdf.txt: 59764 bytes, checksum: 1ebac4580a41f44534b4dad6f32c761e (MD5) Previous issue date: 2006-03-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:13:27Z No. of bitstreams: 2 S1677-04202006000100008.pdf: 927918 bytes, checksum: 9258fae108c1ae62bed73557138b4add (MD5) S1677-04202006000100008.pdf.txt: 59764 bytes, checksum: 1ebac4580a41f44534b4dad6f32c761e (MD5) Made available in DSpace on 2014-05-20T13:13:27Z (GMT). No. of bitstreams: 2 S1677-04202006000100008.pdf: 927918 bytes, checksum: 9258fae108c1ae62bed73557138b4add (MD5) S1677-04202006000100008.pdf.txt: 59764 bytes, checksum: 1ebac4580a41f44534b4dad6f32c761e (MD5) Previous issue date: 2006-03-01 O café é um dos principais produtos agrícolas, sendo considerado o segundo item em importância do comércio internacional de commodities. O gênero Coffea pertence à família Rubiaceae que também inclui outras plantas importantes. Este gênero contém aproximadamente 100 espécies, mas a produção comercial é baseada somente em duas espécies, Coffea arabica e Coffea canephora, que representam aproximadamente 70 % e 30 % do mercado total de café, respectivamente. O Projeto Genoma Café Brasileiro foi desenvolvido com o objetivo de disponibilizar os modernos recursos da genômica à comunidade científica e aos diferentes segmentos da cadeia produtiva do café. Para isso, foram seqüenciados 214.964 clones escolhidos aleatoriamente de 37 bibliotecas de cDNA de C. arabica, C. canephora e C. racemosa representando estádios específicos do desenvolvimento de células e de tecidos do cafeeiro, resultando em 130.792, 12.381 e 10.566 seqüências de cada espécie, respectivamente, após processo de trimagem. Os ESTs foram agrupados em 17.982 contigs e em 32.155 singletons. A comparação destas seqüências pelo programa BLAST revelou que 22 % não tiveram nenhuma similaridade significativa às seqüências no banco de dados do National Center for Biotechnology Information (de função conhecida ou desconhecida). A base de dados de ESTs do cafeeiro resultou na identificação de cerca de 33.000 unigenes diferentes. Os resultados de anotação das seqüências foram armazenados em base de dados online em http://www.lge.ibi.unicamp.br/cafe. Os recursos desenvolvidos por este projeto disponibilizam ferramentas genéticas e genômicas que podem ser decisivas para a sustentabilidade, a competitividade e a futura viabilidade da agroindústria cafeeira nos mercados interno e externo. Coffee is one of the most valuable agricultural commodities and ranks second on international trade exchanges. The genus Coffea belongs to the Rubiaceae family which includes other important plants. The genus contains about 100 species but commercial production is based only on two species, Coffea arabica and Coffea canephora that represent about 70 % and 30 % of the total coffee market, respectively. The Brazilian Coffee Genome Project was designed with the objective of making modern genomics resources available to the coffee scientific community, working on different aspects of the coffee production chain. We have single-pass sequenced a total of 214,964 randomly picked clones from 37 cDNA libraries of C. arabica, C. canephora and C. racemosa, representing specific stages of cells and plant development that after trimming resulted in 130,792, 12,381 and 10,566 sequences for each species, respectively. The ESTs clustered into 17,982 clusters and 32,155 singletons. Blast analysis of these sequences revealed that 22 % had no significant matches to sequences in the National Center for Biotechnology Information database (of known or unknown function). The generated coffee EST database resulted in the identification of close to 33,000 different unigenes. Annotated sequencing results have been stored in an online database at http://www.lge.ibi.unicamp.br/cafe. Resources developed in this project provide genetic and genomic tools that may hold the key to the sustainability, competitiveness and future viability of the coffee industry in local and international markets. IAPAR Laboratório de Biotecnologia Vegetal Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) Recursos Genéticos e Biotecnologia Núcleo de Biotecnologia-NTBio Instituto Agronômico de Campinas USP Escola Superior de Agricultura Luiz de Queiroz Departamento de Genética USP Superior de Agricultura Luiz de Queiroz Departamento de Zootecnia USP Escola Superior de Agricultura Luiz de Queiroz Departamento de Entomologia, Fitopatologia e Zoologia Agrícola USP Escola Superior de Agricultura Luiz de Queiroz Departamento de Ciências Biológicas UNESP Instituto de Biociências Departamento de Genética Universidade Federal de São Carlos (UFSCar) Centro de Ciências Agrárias USP Instituto de Ciências Biomédicas UNESP Faculdade de Ciências Agronômicas Departamento de Defesa Fitossanitária UNESP Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal Departamento de Tecnologia UNESP Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal Departamento de Biologia Aplicada à Agropecuária USP Instituto de Química Departamento de Bioquímica UNICAMP Instituto de Biologia Departamento de Fisiologia Vegetal UNICAMP Faculdade de Engenharia Química Instituto da Computação UNICAMP Instituto da Computação Laboratório de Bioinformática Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) Café IAPAR IAC Centro APTA de Citros Sylvio Moreira USP Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto Departamento de Biologia USP Instituto de Biociências Departamento de Botânica USP Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) Café Instituto Agronômico de Campinas Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) Café Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) Recursos Genéticos e Biotecnologia Núcleo de Biotecnologia-NTBio UNICAMP Centro de Biologia Molecular e Engenharia Genética Universidade de Mogi das Cruzes Núcleo Integrado de Biotecnologia Instituto Biológico de São Paulo Centro de Sanidade Vegetal USP Centro de Energia Nuclear na Agricultura UNICAMP Instituto de Biologia Laboratório de Genômica e Expressão UNESP Instituto de Biociências Departamento de Genética UNESP Faculdade de Ciências Agronômicas Departamento de Defesa Fitossanitária UNESP Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal Departamento de Tecnologia UNESP Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal Departamento de Biologia Aplicada à Agropecuária

Background In higher plants, the inhibition of photosynthetic capacity under drought is attributable to stomatal and non-stomatal (i.e., photochemical and biochemical) effects. In particular, a disruption of photosynthetic metabolism and Rubisco regulation can be observed. Several studies reported reduced expression of the RBCS genes, which encode the Rubisco small subunit, under water stress. Results Expression of the RBCS1 gene was analysed in the allopolyploid context of C. arabica, which originates from a natural cross between the C. canephora and C. eugenioides species. Our study revealed the existence of two homeologous RBCS1 genes in C. arabica: one carried by the C. canephora sub-genome (called CaCc) and the other carried by the C. eugenioides sub-genome (called CaCe). Using specific primer pairs for each homeolog, expression studies revealed that CaCe was expressed in C. eugenioides and C. arabica but was undetectable in C. canephora. On the other hand, CaCc was expressed in C. canephora but almost completely silenced in non-introgressed ("pure") genotypes of C. arabica. However, enhanced CaCc expression was observed in most C. arabica cultivars with introgressed C. canephora genome. In addition, total RBCS1 expression was higher for C. arabica cultivars that had recently introgressed C. canephora genome than for "pure" cultivars. For both species, water stress led to an important decrease in the abundance of RBCS1 transcripts. This was observed for plants grown in either greenhouse or field conditions under severe or moderate drought. However, this reduction of RBCS1 gene expression was not accompanied by a decrease in the corresponding protein in the leaves of C. canephora subjected to water withdrawal. In that case, the amount of RBCS1 was even higher under drought than under unstressed (irrigated) conditions, which suggests great stability of RBCS1 under adverse water conditions. On the other hand, for C. arabica, high nocturnal expression of RBCS1 could also explain the accumulation of the RBCS1 protein under water stress. Altogether, the results presented here suggest that the content of RBCS was not responsible for the loss of photosynthetic capacity that is commonly observed in water-stressed coffee plants. Conclusion We showed that the CaCe homeolog was expressed in C. eugenioides and non-introgressed ("pure") genotypes of C. arabica but that it was undetectable in C. canephora. On the other hand, the CaCc homeolog was expressed in C. canephora but highly repressed in C. arabica. Expression of the CaCc homeolog was enhanced in C. arabica cultivars that experienced recent introgression with C. canephora. For both C. canephora and C. arabica species, total RBCS1 gene expression was highly reduced with WS. Unexpectedly, the accumulation of RBCS1 protein was observed in the leaves of C. canephora under WS, possibly coming from nocturnal RBCS1 expression. These results suggest that the increase in the amount of RBCS1 protein could contribute to the antioxidative function of photorespiration in water-stressed coffee plants.

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