Felipe Vinecky

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.

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.

Selection of reliable reference genes for RT-qPCR analysis during developmental stages and abiotic stress in Setaria viridis.

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.

Post-secretory events alter the peptide content of the skin secretion of Hypsiboas raniceps.

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.

Suppression of a single BAHD gene in Setaria viridis causes large, stable decreases in cell wall feruloylation and increases biomass digestibility

Summary 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.

Overexpression of BdMATE Gene Improves Aluminum Tolerance in Setaria viridis

Acidic soils are distributed worldwide, predominantly in tropical and subtropical areas, reaching around 50% of the arable soil. This type of soil strongly reduces crop production, mainly because of the presence of aluminum, which has its solubility increased at low pH levels. A well-known physiological mechanism used by plants to cope with Al stress involves activation of membrane transporters responsible for organic acid anions secretion from the root apex to the rhizosphere, which chelate Al, preventing its absorption by roots. In sorghum, a membrane transporter gene belonging to multidrug and toxic compound extrusion (MATE) family was identified and characterized as an aluminum-activated citrate transporter gene responsible for Al tolerance in this crop. Setaria viridis is an emerging model for C4 species and it is an important model to validate some genes for further C4 crops transformation, such as sugarcane, maize, and wheat. In the present work, Setaria viridis was used as a model plant to overexpress a newly identified MATE gene from Brachypodium distachyon (BdMATE), closely related to SbMATE, for aluminum tolerance assays. Transgenic S. viridis plants overexpressing a BdMATE presented an improved Al tolerance phenotype, characterized by sustained root growth and exclusion of aluminum from the root apex in transgenic plants, as confirmed by hematoxylin assay. In addition, transgenic plants showed higher root citrate exudation into the rhizosphere, suggesting that Al tolerance improvement in these plants could be related to the chelation of the metal by the organic acid anion. These results suggest that BdMATE gene can be used to transform C4 crops of economic importance with improved aluminum tolerance.

Characterization of sugarcane (Saccharum spp.) leaf senescence: implications for biofuel production

BackgroundSecond-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.ResultsThe 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.ConclusionsOur 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.

Nucleotide Diversity of the Coding and Promoter Regions of DREB1D, a Candidate Gene for Drought Tolerance in Coffea Species

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.