Luiz Gonzaga Esteves Vieira

Molecular mapping of soybean rust ( Phakopsora pachyrhizi ) resistance genes: discovery of a novel locus and alleles

Soybean production in South and North America has recently been threatened by the widespread dissemination of soybean rust (SBR) caused by the fungus Phakopsora pachyrhizi. Currently, chemical spray containing fungicides is the only effective method to control the disease. This strategy increases production costs and exposes the environment to higher levels of fungicides. As a first step towards the development of SBR resistant cultivars, we studied the genetic basis of SBR resistance in five F2 populations derived from crossing the Brazilian-adapted susceptible cultivar CD 208 to each of five different plant introductions (PI 200487, PI 200526, PI 230970, PI 459025, PI 471904) carrying SBR-resistant genes (Rpp). Molecular mapping of SBR-resistance genes was performed in three of these PIs (PI 459025, PI 200526, PI 471904), and also in two other PIs (PI 200456 and 224270). The strategy mapped two genes present in PI 230970 and PI 459025, the original sources of Rpp2 and Rpp4, to linkage groups (LG) J and G, respectively. A new SBR resistance locus, rpp5 was mapped in the LG-N. Together, the genetic and molecular analysis suggested multiple alleles or closely linked genes that govern SBR resistance in soybean.

Brazilian coffee genome project: an EST-based genomic resource

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.

Expression of three galactinol synthase isoforms in Coffea arabica L. and accumulation of raffinose and stachyose in response to abiotic stresses.

Galactinol synthase (EC 2.4.1.123; GolS) catalyzes the first step in the synthesis of raffinose family oligosaccharides (RFOs). Their accumulation in response to abiotic stresses implies a role for RFOs in stress adaptation. In this study, the expression patterns of three isoforms of galactinol synthase (CaGolS1-2-3) from Coffea arabica were evaluated in response to water deficit, salinity and heat stress. All CaGolS isoforms were highly expressed in leaves while little to no expression were detected in flower buds, flowers, plagiotropic shoots, roots, endosperm and pericarp of mature fruits. Transcriptional analysis indicated that the genes were differentially regulated under water deficit, high salt and heat stress. CaGolS1 isoform is constitutively expressed in plants under normal growth conditions and was the most responsive during all stress treatments. CaGolS2 is unique among the three isoforms in that it was detected only under severe water deficit and salt stresses. CaGolS3 was primarily expressed under moderate and severe drought. This isoform was induced only at the third day of heat and under high salt stress. The increase in GolS transcription was not reflected into the amount of galactinol in coffee leaves, as specific glycosyltransferases most likely used galactinol to transfer galactose units to higher homologous oligosaccharides, as suggested by the increase of raffinose and stachyose during the stresses.

The accumulation of endogenous proline induces changes in gene expression of several antioxidant enzymes in leaves of transgenic Swingle citrumelo

Plant exposure to abiotic stresses leads to an accumulation of reactive oxygen species with the concomitant increase in antioxidant defense mechanisms. Previous studies showed that exogenous application of proline mitigate the deleterious effects caused by oxidative stress due to its ability to increase the activity of antioxidant enzymes. However, there are no reports of the effects of high endogenous accumulation of proline in the transcriptional pattern of antioxidant enzymes genes under normal conditions of water supply or in response to water deficit. Here, we show that isoforms of four antioxidant enzymes genes (Ascorbate peroxidase—APX, Catalase—CAT, Superoxide dismutase—SOD and Glutathione reductase—GR) were differentially regulated in leaves of Swingle citrumelo transgenic plants with high endogenous proline accumulation submitted to water deficits and also under normal water supply condition. Proline per se caused a two-fold change in the transcription activity of APX1, APXcl, CAT2 and Cu/ZnSOD2, while during water deficit proline influenced mRNAs levels in APXs and Cu/ZnSODs isoforms, MnSODmit and GRcl. This study adds new information on the role of proline during drought conditions and, more important, without the potential confounding effects imposed by water deficiency. We showed that, in addition to its known effects on diverse plant physiological and biochemical processes, high endogenous proline can also acts as a regulatory/signalling molecule capable of altering the transcript levels of stress-related genes.

Differentially expressed genes and proteins upon drought acclimation in tolerant and sensitive genotypes of Coffea canephora

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 High-Throughput Data Mining of Single Nucleotide Polymorphisms in Coffea Species Expressed Sequence Tags Suggests Differential Homeologous Gene Expression in the Allotetraploid Coffea arabica

Polyploidization constitutes a common mode of evolution in flowering plants. This event provides the raw material for the divergence of function in homeologous genes, leading to phenotypic novelty that can contribute to the success of polyploids in nature or their selection for use in agriculture. Mounting evidence underlined the existence of homeologous expression biases in polyploid genomes; however, strategies to analyze such transcriptome regulation remained scarce. Important factors regarding homeologous expression biases remain to be explored, such as whether this phenomenon influences specific genes, how paralogs are affected by genome doubling, and what is the importance of the variability of homeologous expression bias to genotype differences. This study reports the expressed sequence tag assembly of the allopolyploid Coffea arabica and one of its direct ancestors, Coffea canephora. The assembly was used for the discovery of single nucleotide polymorphisms through the identification of high-quality discrepancies in overlapped expressed sequence tags and for gene expression information indirectly estimated by the transcript redundancy. Sequence diversity profiles were evaluated within C. arabica (Ca) and C. canephora (Cc) and used to deduce the transcript contribution of the Coffea eugenioides (Ce) ancestor. The assignment of the C. arabica haplotypes to the C. canephora (CaCc) or C. eugenioides (CaCe) ancestral genomes allowed us to analyze gene expression contributions of each subgenome in C. arabica. In silico data were validated by the quantitative polymerase chain reaction and allele-specific combination TaqMAMA-based method. The presence of differential expression of C. arabica homeologous genes and its implications in coffee gene expression, ontology, and physiology are discussed.

Evaluation of kahweol and cafestol in coffee tissues and roasted coffee by a new high-performance liquid chromatography methodology.

A reverse phase high-performance liquid chromatography (HPLC) method was developed for the simultaneous quantification of kahweol and cafestol in tissues of fresh fruits, leaves, and roasted coffee beans. The best resolution was obtained with isocratic elution of acetonitrile/water (55/45% v/v) and UV detection. A single sample preparation method carried out by direct saponification and extraction with organic solvent was standardized for all matrices. Good recovery (average of 99% for kahweol and 94% for cafestol), repeatability, and linearity were obtained. Detection limits of 2.3 and 3.0 mg/100 g were observed for kahweol and cafestol. The HPLC method was effective in quantifying these diterpenes in the different coffee matrices. The endosperm and perisperm of Coffea arabica cv. IAPAR 59 showed elevated amounts of kahweol as compared to the pericarp and leaves. On the other hand, cafestol was detected in all samples except in leaves from Coffea canephora cv. Apoatā.

Effects of shade on the development and sugar metabolism of coffee (Coffea arabica L.) fruits.

Coffee fruits grown in shade are characterized by larger bean size than those grown under full-sun conditions. The present study assessed the effects of shade on bean characteristics and sugar metabolism by analyzing tissue development, sugar contents, activities of sucrose metabolizing enzymes and expression of sucrose synthase-encoding genes in fruits of coffee (Coffea arabica L.) plants submitted to full-sun (FS) and shade (SH) conditions. Evolution of tissue fresh weights measured in fruits collected regularly from flowering to maturation indicated that this increase is due to greater development of the perisperm tissue in the shade. The effects of light regime on sucrose and reducing sugar (glucose and fructose) contents were studied in fresh and dry coffee beans. Shade led to a significant reduction in sucrose content and to an increase in reducing sugars. In pericarp and perisperm tissues, higher activities of sucrose synthase (EC 2.4.1.13) and sucrose-phosphate synthase (SPS: EC 2.4.1.14) were detected at maturation in the shade compared with full sun. These two enzymes also had higher peaks of activities in developing endosperm under shade than in full sun. It was also noted that shade modified the expression of SUS-encoding genes in coffee beans; CaSUS2 gene transcripts levels were higher in SH than in FS. As no sucrose increase accompanied these changes, this suggests that sucrose metabolism was redirected to other metabolic pathways that need to be identified.

Ethylene production and acc oxidase gene expression during fruit ripening of Coffea arabica L.

The phytohormone ethylene is involved in several physiological and developmental processes in higher plants, including ripening of fruits, abscission of organs and tissues, senescence, wound response as well as in other abiotic stresses. The enzyme 1-aminocyclopropane-1-carboxylate oxidase (ACO) catalyzes the last step of ethylene biosynthesis. The production of ethylene and the expression of a Coffea arabica ACO gene during the last stages of fruit maturation were investigated. A rapid increase of ethylene production at the green-yellow stage of fruit ripening, after the end of endosperm formation, and a decrease of ethylene production at the cherry stage indicates a climacteric phase during ripening. An ACC oxidase (Ca-ACO) from coffee fruit cDNA was cloned and characterized using primers previously reported. The cDNA is homologous to previously described ACC oxidase cDNA in Coffea. The nucleotide and amino acid deduced sequences of the clone showed high homology with ACO from climacteric fruits. Northern blots were performed to determine the Ca-ACO transcription pattern from different tissues and from fruits at different ripening stages. Coffee fruits at an early ripening stage (green) showed the lowest level of Ca-ACO transcript accumulation. The transcript levels of Ca-ACO did not change significantly during the later stages, suggesting the presence of post- transcriptional control mechanisms. These results, taken together, strongly suggest a climacteric nature of coffee fruit ripening.

Nitrogen Starvation, Salt and Heat Stress in Coffee (Coffea arabica L.): Identification and Validation of New Genes for qPCR Normalization

Abiotic stresses are among the most important factors that affect food production. One important step to face these environmental challenges is the transcriptional modulation. Quantitative real-time PCR is a rapid, sensitive, and reliable method for the detection of mRNAs and it has become a powerful tool to mitigate plant stress tolerance; however, suitable reference genes are required for data normalization. Reference genes for coffee plants during nitrogen starvation, salinity and heat stress have not yet been reported. We evaluated the expression stability of ten candidate reference genes using geNorm PLUS, NormFinder, and BestKeeper softwares, in plants submitted to nitrogen starvation, salt and heat stress. EF1, EF1α, GAPDH, MDH, and UBQ10 were ranked as the most stable genes in all stresses and software analyses, while RPL39 and RPII were classified as the less reliable references. For reference gene validation, the transcriptional pattern of a Coffea non-symbiotic hemoglobin (CaHb1) was analyzed using the two new recommended and the most unstable gene references for normalization. The most unstable gene may lead to incorrect interpretation of CaHb1 transcriptional analysis. Here, we recommend two new reference genes in Coffea for use in data normalization in abiotic stresses: MDH and EF1.