Paula Macedo Nobile

Evaluation of coffee reference genes for relative expression studies by quantitative real-time RT-PCR

Accuracy in quantitative real-time polymerase chain reaction (qPCR) requires the use of stable endogenous controls. Normalization with multiple reference genes is the gold standard, but their identification is a laborious task, especially in species with limited sequence information. Coffee (Coffea ssp.) is an important agricultural commodity and, due to its economic relevance, is the subject of increasing research in genetics and biotechnology, in which gene expression analysis is one of the most important fields. Notwithstanding, relatively few works have focused on the analysis of gene expression in coffee. Moreover, most of these works have used less accurate techniques such as northern blot assays instead of more accurate techniques (e.g., qPCR) that have already been extensively used in other plant species. Aiming to boost the use of qPCR in studies of gene expression in coffee, we uncovered reference genes to be used in a number of different experimental conditions. Using two distinct algorithms implemented by geNorm and Norm Finder, we evaluated a total of eight candidate reference genes (psaB, PP2A, AP47, S24, GAPDH, rpl39, UBQ10, and UBI9) in four different experimental sets (control versus drought-stressed leaves, control versus drought-stressed roots, leaves of three different coffee cultivars, and four different coffee organs). The most suitable combination of reference genes was indicated in each experimental set for use as internal control for reliable qPCR data normalization. This study also provides useful guidelines for reference gene selection for researchers working with coffee plant samples under conditions other than those tested here.

Lignification in Sugarcane: Biochemical Characterization, Gene Discovery, and Expression Analysis in Two Genotypes Contrasting for Lignin Content

Biochemical, histological, and transcriptional characterization of lignification identifies substantial differences in two sugarcane genotypes. Sugarcane (Saccharum spp.) is currently one of the most efficient crops in the production of first-generation biofuels. However, the bagasse represents an additional abundant lignocellulosic resource that has the potential to increase the ethanol production per plant. To achieve a more efficient conversion of bagasse into ethanol, a better understanding of the main factors affecting biomass recalcitrance is needed. Because several studies have shown a negative effect of lignin on saccharification yield, the characterization of lignin biosynthesis, structure, and deposition in sugarcane is an important goal. Here, we present, to our knowledge, the first systematic study of lignin deposition during sugarcane stem development, using histological, biochemical, and transcriptional data derived from two sugarcane genotypes with contrasting lignin contents. Lignin amount and composition were determined in rind (outer) and pith (inner) tissues throughout stem development. In addition, the phenolic metabolome was analyzed by ultra-high-performance liquid chromatography-mass spectrometry, which allowed the identification of 35 compounds related to the phenylpropanoid pathway and monolignol biosynthesis. Furthermore, the Sugarcane EST Database was extensively surveyed to identify lignin biosynthetic gene homologs, and the expression of all identified genes during stem development was determined by quantitative reverse transcription-polymerase chain reaction. Our data provide, to our knowledge, the first in-depth characterization of lignin biosynthesis in sugarcane and form the baseline for the rational metabolic engineering of sugarcane feedstock for bioenergy purposes.

Expression Profile of Sugarcane Transcription Factor Genes Involved in Lignin Biosynthesis

Cell wall recalcitrance, which is conferred in part by lignin, is the main bottleneck in lignocellulosic ethanol production. Transcription factors (TFs) have been suggested as targets to reduce or modify lignin. Here we analysed the expression profile of nine sugarcane TFs, their relationships with genes of the monolignol biosynthesis pathway, and their effects on lignin content and composition. Our assays compared two sugarcane genotypes with different lignin contents. To identify differences between tissue types and between the top and bottom of the plant, the culm was divided into intermediary and mature internodes, and the internodes were separated into pith and rind. The expression profiles obtained for the nine TFs were rather complex, showing that not only the genotype but also the tissue type and developmental stage influenced the results. Pearson correlation analysis indicated that ShMYB58/63 was positively correlated with the syringyl/guaiacyl ratio. In addition, a Bayesian network showed predicted interactions between the TFs and genes for lignin biosynthesis that were previously reported in the literature, as well as novel interactions such as those between ShMYB58/63 and ShF5H. These findings suggest that in sugarcane culm, the differential lignin deposition between tissue types (rind and pith) and at different developmental stages is under transcriptional regulation.

Influence of air temperature on proteinase activity and beverage quality in Coffea arabica

Fruits were collected from trees of Coffea arabica cv. Obata grown at Mococa and Adamantina in Sao Paulo State, Brazil, which are regions with marked differences in air temperature that produce coffee with distinct qualities. Mococa is a cooler location that produces high-quality coffee, whereas coffee from Adamantina is of lower quality. The amino acid and protein contents, amino acid profile, and proteinase activity and type in endosperm protein extracts were analysed. Proteinase genes were identified, and their expression was assayed. All results indicate that temperature plays a role in controlling proteinase activity in coffee endosperm. Proteinase activity was higher in the endosperm of immature fruits from Adamantina, which was correlated with higher amino acid content, changes in the amino acid profile, and increased gene expression. Cysteine proteinases were the main class of proteinases in the protein extracts. These data suggest that temperature plays an important role in coffee quality by altering nitrogen compound composition.

Identification, classification and transcriptional profiles of dirigent domain-containing proteins in sugarcane

Dirigent (DIR) proteins, encoded by DIR genes, are referred to as “dirigent” because they direct the outcome of the coupling of the monolignol coniferyl alcohol into (+) or (−) pinoresinol, the first intermediates in the enantiocomplementary pathways for lignan biosynthesis. DIR domain-containing or DIR-like proteins are, thus, termed for not having a clear characterization. A transcriptome- and genome-wide survey of DIR domain-containing proteins in sugarcane was carried out, in addition to phylogenetic, physicochemical and transcriptional analyses. A total of 120 non-redundant sequences containing the DIR domain were identified and classified into 64 groups according to phylogenetic and sequence alignment analyses. In silico analysis of transcript abundance showed that these sequences are expressed at low levels in leaves and genes in the same phylogenetic clade have similar expression patterns. Expression analysis of ShDIR1-like transcripts in the culm internodes of sugarcane demonstrates their abundance in mature internodes, their induction by nitrogen fertilization and their predominant expression in cells that have a lignified secondary cell wall, such as vascular bundles of young internodes and parenchymal cells of the pith of mature internodes. Due to the lack of information about the functional role of DIR in plants, a possible relationship is discussed between the ShDIR1-like transcriptional profile and cell wall development in parenchyma cells of sugarcane culm, which typically accumulates large amounts of sucrose. The number of genes encoding the DIR domain-containing proteins in sugarcane is intriguing and is an indication per se that these proteins may have an important metabolic role and thus deserve to be better studied.

Antioxidative responses of cell suspension cultures of two Coffea arabica varieties to low aluminum levels at pH 5.8

The effects of aluminum (Al) on the activities of antioxidant enzymes and ferritin expression were studied in cell suspension cultures of two varieties of Coffea arabica, Mundo Novo and Icatu, in medium with pH at 5.8. The cells were incubated with 300 µM Al3+, and the Al speciation as Al3+ was 1.45% of the mole fraction. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) were increased in Mundo Novo, whereas glutathione reductase (GR) and guaiacol peroxidase (GPOX) activities remained unchanged. SOD, GR, and GST activities were increased in Icatu, while CAT activity was not changed, and GPOX activity decreased. The expression of two ferritin genes (CaFer1 and CaFer2) were analyzed by Real-Time PCR. Al caused a downregulation of CaFER1 expression and no changes of CaFER2 expression in both varieties. The Western blot showed no alteration in ferritin protein levels in Mundo Novo and a decrease in Icatu. The differential enzymes responses indicate that the response to Al is variety-dependent.

Transcriptional profile of genes involved in the biosynthesis of phytate and ferritin in Coffea.

The present work aimed to study the control of the biosynthesis of the antinutritional factor phytate and its associated Fe-rich protein family, ferritin, in coffee. Phytate has the ability to chelate Fe, making it unavailable to human absorption. The Coffea genome databases were queried for genes associated with phytate metabolism and ferritin genes. The genetic framework for phytate biosynthesis and its reverse pathway was identified in silico analyses and indicate that Coffea phosphatidyl inositol kinase and monophosphatase families play nonredundant roles in phytate metabolism. The transcriptional profiles of phytate biosynthesis key-genes MYO-INOSITOL(3)P1 SYNTHASE, two genes coding for PHOSPHATIDYL INOSITOL KINASE, and three FERRITIN genes were temporally evaluated by qPCR in coffee seeds from two crop locations, Adamantina-SP and Ouro-Fino-MG, the last one traditionally associated with high-quality coffee beverage grain. A targeted metabolome profile of phytic acid contents throughout three fruit maturation stages in association with the transcriptional analysis was also obtained. Taken together, our data indicate that the investigated local conditions did not cause significant alterations in phytate biosynthesis. Futhermore, the temporal transcriptional profiling revealed that candidate gene expression is regulated independently of phytate accumulation. In contrast, the expression profile of ferritin-unit genes is affected by environmental conditions and genetic background. The roles of the investigated genes are discussed concerning the quality of coffee beverage.

Characterization, isolation and cloning of sugarcane genes related to drought stress

Background Sugarcane is a major crop worldwide as raw material for sugar and ethanol production. Drought is one of the most limiting factor that affects sugarcane productivity. In order to understand the mechanisms of drought response, field and greenhouse assays were conducted with two droughtcontrasting sugarcane genotypes (IACSP94-2094/tolerant and IACSP97-7065/sensitive), and several genes up/downregulated under drought stress identified by microarrays and RNAseq analyses. Ten differential expressed genes in both assays were evaluated by qPCR [1], and three of them showed the transcriptional profile related drought tolerance: aLipoxygenase (ScLOX), acting in the biosynthesis of the jasmonic acid precursor, and recent studies showed their role in defense against drought stress [3]; bDehydrin, correlated to drought stress and associated to maintenance of turgor cells, [2]; cDirigent-jacalin, associated to resistance disease and abiotic stress tolerance [4] and also related to jasmonic acid, an important hormone on plant defense. These genes were chosen as target for functional analyses in rice and sugarcane transgenic plants.

In situ localization of mRNA of resembling the dirigent protein in sugarcane stems

Background The dirigent proteins (DP) families and resembling the DP (DP-like) are exclusive in land plants and related to plants defense and forming phenylpropanoids compounds optically active, mainly pinoresinol [1]. Pinoresinol is converted into a variety of lignans. The dirigent protein involvement is not confirmed in lignin formation, a biopolymer which is a negative factor for the ethanol cellulosic production. However, recently it was demonstrated Arabidopsis DP-like, AtDIR10, localization in the lignin polymerization site and a determinant role in the formation of a lignin specific root structure, named as Casparian strip [2]. Despite of the controversy of the DP forming-lignin its substrate diversity is consistent. Since DPs and DPs-like are represented by numerous members of gene families with high diversified sequences and with unknown functional role for most of them [3]. One of the sugarcane DP-like, named as ShDP1-like, showed special interesting due its high level of expression in the pith of mature stem [3] coinciding with higher level of the sinapyl (S) unit forming lignin [4]. Localization of DP expression to particular cell and tissue types is a necessary prerequisite in understanding the biological role of this gene.