Genomic and transcriptomic inventory of membrane transporters in coffee: Exploring molecular mechanisms of metabolite accumulation.

The genus Coffea (Rubiaceae) encompasses a group of perennial plant species, including a commodity crop from which seeds are roasted, ground, and infused to make one of the most appreciated beverages in the world. As an important tropical crop restricted to specific regions of the world, coffee production is highly susceptible to the effects of environmental instabilities (i.e., local year-to-year weather fluctuations and global climate change) and threatening pest pressures, not to mention an increasing quality rigor by consumers in industrialized countries. Specialized metabolites are substances that largely affect plant-environment interactions as well as how consumers experience agricultural products. Membrane transporters are key targets, albeit understudied, for understanding and tailoring the spatiotemporal distribution of specialized metabolites as they mediate and control molecular trafficking and substance accumulation. Therefore, we analyzed the transportome of C. canephora encoded within the 25,574 protein-coding genes annotated in the genome of this species and identified 1847 putative membrane transporters. Following, we mined 152 transcriptional profiles of C. canephora and C. arabica and performed a comprehensive co-expression analysis to identify transporters potentially involved in the accumulation of specialized metabolites associated with beverage quality and bioactivity attributes. In toto, this report points to an avenue of possibilities on Coffea genomic and transcriptomic data mining for genetic breeding strategies, which can lead to the development of new, resilient varieties for more sustainable coffee production systems.

Gene Expression Profile Analysis is Directly Affected by the Selected Reference Gene: The Case of Leaf-Cutting Atta Sexdens.

Although several ant species are important targets for the development of molecular control strategies, only a few studies focus on identifying and validating reference genes for quantitative reverse transcription polymerase chain reaction (RT-qPCR) data normalization. We provide here an extensive study to identify and validate suitable reference genes for gene expression analysis in the ant Atta sexdens, a threatening agricultural pest in South America. The optimal number of reference genes varies according to each sample and the result generated by RefFinder differed about which is the most suitable reference gene. Results suggest that the RPS16, NADH and SDHB genes were the best reference genes in the sample pool according to stability values. The SNF7 gene expression pattern was stable in all evaluated sample set. In contrast, when using less stable reference genes for normalization a large variability in SNF7 gene expression was recorded. There is no universal reference gene suitable for all conditions under analysis, since these genes can also participate in different cellular functions, thus requiring a systematic validation of possible reference genes for each specific condition. The choice of reference genes on SNF7 gene normalization confirmed that unstable reference genes might drastically change the expression profile analysis of target candidate genes.

Subspecies and diet affect the expression of genes involved in lipid metabolism and chemical composition of muscle in beef cattle.

Fourteen Nellore and 14 Angus young bulls with BW of 381±11.8kg were randomly assigned into 2 feeding groups (whole shelled corn without forage (WSC) or corn silage and ground corn (GC)) to evaluate chemical composition and expression of genes involved in lipid metabolism in the longissimus thoracis (LT). We hypothesized that bulls fed the WSC diet have greater amounts of intramuscular fat and Angus have higher expression levels of PPAR and SREBF. Meat from Angus bulls had greater ether extract compared to Nellore (P<0.05). Muscle from bulls fed the WSC diet had greater expression of PPARA (P<0.05) and lower levels of SREBF1 expression (P<0.01). The LT of Nellore fed GC had greater expression of FABP4, ACACA and SCD genes (P<0.01). In conclusion, the greater concentration of starch in the WSC diet did not increase marbling in the beef of bulls fed this diet due to the reduced expression of SREBF1.

Molecular epidemiology of Streptococcus agalactiae isolated from mastitis in Brazilian dairy herds

Abstract Streptococcus agalactiae is one of the most common pathogens leading to mastitis in dairy herds worldwide; consequently, the pathogen causes major economic losses for affected farmers. In this study, multilocus sequence typing (MLST), genotypic capsular typing by multiplex polymerase chain reaction (PCR), and virulence gene detection were performed to address the molecular epidemiology of 59 bovine (mastitis) S. agalactiae isolates from 36 dairy farms located in the largest milk-producing mesoregions in Brazil (Minas Gerais, São Paulo, Paraná, and Pernambuco). We screened for the virulence genes bac, bca, bibA, cfb, hylB, fbsA, fbsB, PI-1, PI-2a, and PI-2b, which are associated with adhesion, invasion, tissue damage, and/or immune evasion. Furthermore, five capsular types were identified (Ia, Ib, II, III, and IV), and a few isolates were classified as non-typeable (NT). MLST revealed the following eight sequence types (STs): ST-61, ST-67, ST-103, ST-146, ST-226, ST-314, and ST-570, which were clustered in five clonal complexes (CC64, CC67, CC103, CC17, and CC314), and one singleton, ST-91. Among the virulence genes screened in this study, PI-2b, fbsB, cfb, and hylB appear to be the most important during mastitis development in cattle. Collectively, these results establish the molecular epidemiology of S. agalactiae isolated from cows in Brazilian herds. We believe that the data presented here provide a foundation for future research aimed at developing and implementing new preventative and treatment options for mastitis caused by S. agalactiae.

Molecular epidemiology of Streptococcus agalactiae isolated from mastitis in Brazilian dairy herds.

Streptococcus agalactiae is one of the most common pathogens leading to mastitis in dairy herds worldwide; consequently, the pathogen causes major economic losses for affected farmers. In this study, multilocus sequence typing (MLST), genotypic capsular typing by multiplex polymerase chain reaction (PCR), and virulence gene detection were performed to address the molecular epidemiology of 59 bovine (mastitis) S. agalactiae isolates from 36 dairy farms located in the largest milk-producing mesoregions in Brazil (Minas Gerais, São Paulo, Paraná, and Pernambuco). We screened for the virulence genes bac, bca, bibA, cfb, hylB, fbsA, fbsB, PI-1, PI-2a, and PI-2b, which are associated with adhesion, invasion, tissue damage, and/or immune evasion. Furthermore, five capsular types were identified (Ia, Ib, II, III, and IV), and a few isolates were classified as non-typeable (NT). MLST revealed the following eight sequence types (STs): ST-61, ST-67, ST-103, ST-146, ST-226, ST-314, and ST-570, which were clustered in five clonal complexes (CC64, CC67, CC103, CC17, and CC314), and one singleton, ST-91. Among the virulence genes screened in this study, PI-2b, fbsB, cfb, and hylB appear to be the most important during mastitis development in cattle. Collectively, these results establish the molecular epidemiology of S. agalactiae isolated from cows in Brazilian herds. We believe that the data presented here provide a foundation for future research aimed at developing and implementing new preventative and treatment options for mastitis caused by S. agalactiae.

The proteome profile of embryogenic cell suspensions of Coffea arabica L.

Somatic embryogenesis, is a process by which new viable embryos are produced from somatic tissues. Somatic embryogenesis is not only a useful biotechnological tool for the massive clonal propagation and genetic engineering but it also allows to obtain fundamental knowledge about the molecular changes that take place during embryogenesis. We present the proteome profile of two embryogenic cell suspensions. We identified 1052 non-redundant proteins. We present their known GO annotations and show two protein networks sharing the GO annotations related to stress and embryogenic capacity via the free program Cytoscape. To our knowledge these results give the first high-throughput proteome description of embryogenic cell suspensions and provide new information about somatic embryos for the whole plant community. The published proteome is a first step toward understanding somatic embryogenesis in coffee and toward a better annotation of proteins in an important non-model crop. All data are available via ProteomeXchange with identifier PXD002963.

Identification and expression analysis of ethylene biosynthesis and signaling genes provides insights into the early and late coffee cultivars ripening pathway.

The plant hormone ethylene is involved in the regulation of a multitude of plant processes, ranging from seed germination to organ senescence. Ethylene induces fruit ripening in climacteric fruits, such as coffee, being directly involved in fruit ripening time and synchronization. Coffee early cultivars usually show a more uniform ripening process although little is known about the genetic factors that promote the earliness of ripening. Thus, this work aimed to characterize the putative members of the coffee (Coffea arabica) ethylene biosynthesis and signaling pathways, as well as to analyze the expression patterns of these members during fruit ripening of early (Catucaí 785-15) and late (Acauã) coffee cultivars. Reverse Transcription-qPCR analysis of the four biosynthesis genes (CaACS1-like; CaACO1-like; CaACO4-like e CaACO5-like) analyzed in this study showed that CaACO1-like and CaACO4-like displayed an expression pattern typically observed in climacteric fruits, being up-regulated during ripening. CaACS1-like gene expression was also up-regulated during fruit ripening of both cultivars, although in a much lesser extent when compared to the changes in CaACO1-like and CaACO4-like gene expression. CaACO5-like was only induced in raisin fruit and may be related to senescence processes. On the other hand, members of the ethylene signaling pathway (CaETR1-like, CaETR4-like, CaCTR2-like, CaEIN2-like, CaEIN3-like, CaERF1) showed slightly higher expression levels during the initial stages of development (green and yellow-green fruits), except for the ethylene receptors CaETR1-like and CaETR4-like, which were constitutively expressed and induced in cherry fruits, respectively. The higher ethylene production levels in Catucaí 785-15 fruits, indicated by the expression analysis of CaACO1-like and CaACO4-like, suggest that it promotes an enhanced CaETR4-like degradation, leading to an increase in ethylene sensitivity and consequently to an earliness in the ripening process of this cultivar. Ethylene production in Acauã fruits may not be sufficient to inactivate the CaETR4-like levels and thus ripening changes occur in a slower pace. Thus, the expression analysis of the ethylene biosynthesis and signaling genes suggests that ethylene is directly involved in the determination of the ripening time of coffee fruits, and CaACO1-like, CaACO4-like and CaETR4-like may display essential roles during coffee fruit ripening.

Induction and characterization of oil palm (Elaeis guineensis Jacq.) pro-embryogenic masses.

Oil palm is one of the most economically valuable oil seed plants, but the expansion of plantations has been limited by availability of seedlings, as the conventional propagation is through seeds, which have low germination rates. One possible solution for the large-scale production is the use of somatic embryogenesis. The aim of this study was evaluate the effects auxins 2,4-D and picloram on the induction of pro-embryogenic masses in E.guineenesis hybrid leaf explants and characterize, regarding embryogenic characteristics, with cytochemical and ultrastructural analysis. Specifically, in vitro plantlets leaves fragments were inoculated in Y3 culture medium supplemented by 2.4-D or picloram at different concentrations (0.0, 1.0, 3.0, 6.0 and 9.0 mg l⁻¹). After 90 days the presence/ absence of cell masses were evaluated. Both growth regulators efficiently induced cellular masses regardless of the concentrations applied. As the cell masses were not homogeneously formed, they were classified according to color and shape into four types: TYPE 1--elongated and translucent, TYPE 2--uneven and translucent, TYPE 3--globular and beige, TYPE 4--globular and white. Based on the anatomical and ultrastructural features, TYPE 2, 3 and 4 cell masses were considered to have the highest embryogenic potential and therefore may be most suited to large-scale vegetative propagation of oil palm.

Evidence for reductive genome evolution and lateral acquisition of virulence functions in two Corynebacterium pseudotuberculosis strains.

BACKGROUND: Corynebacterium pseudotuberculosis, a gram-positive, facultative intracellular pathogen, is the etiologic agent of the disease known as caseous lymphadenitis (CL). CL mainly affects small ruminants, such as goats and sheep; it also causes infections in humans, though rarely. This species is distributed worldwide, but it has the most serious economic impact in Oceania, Africa and South America. Although C. pseudotuberculosis causes major health and productivity problems for livestock, little is known about the molecular basis of its pathogenicity. METHODOLOGY AND FINDINGS: We characterized two C. pseudotuberculosis genomes (Cp1002, isolated from goats; and CpC231, isolated from sheep). Analysis of the predicted genomes showed high similarity in genomic architecture, gene content and genetic order. When C. pseudotuberculosis was compared with other Corynebacterium species, it became evident that this pathogenic species has lost numerous genes, resulting in one of the smallest genomes in the genus. Other differences that could be part of the adaptation to pathogenicity include a lower GC content, of about 52%, and a reduced gene repertoire. The C. pseudotuberculosis genome also includes seven putative pathogenicity islands, which contain several classical virulence factors, including genes for fimbrial subunits, adhesion factors, iron uptake and secreted toxins. Additionally, all of the virulence factors in the islands have characteristics that indicate horizontal transfer. CONCLUSIONS: These particular genome characteristics of C. pseudotuberculosis, as well as its acquired virulence factors in pathogenicity islands, provide evidence of its lifestyle and of the pathogenicity pathways used by this pathogen in the infection process. All genomes cited in this study are available in the NCBI Genbank database (http://www.ncbi.nlm.nih.gov/genbank/) under accession numbers CP001809 and CP001829.

PR gene families of citrus: their organ specific-biotic and abiotic inducible expression profiles based on ESTs approach

In silico expression profiles, of the discovered 3,103 citrus ESTs putatively encoding for PR protein families (PR-1 to PR-17), were evaluated using the Brazil citrus genome EST CitEST/database. Hierarchical clustering was displayed to identify similarities in expression patterns among citrus PR-like gene families (PRlgf) in 33 selected cDNA libraries. In this way, PRlgf preferentially expressed by organ and citrus species, and library conditions were highlighted. Changes in expression profiles of clusters for each of the 17 PRlgf expressed in organs infected by pathogens or drought-stressed citrus species were displayed for relative suppression or induction gene expression in relation to the counterpart control. Overall, few PRlgf showed expression 2-fold higher in pathogen-infected than in uninfected organs, even though the differential expression profiles displayed have been quite diverse among studied species and organs. Furthermore, an insight into some contigs from four PRlgf pointed out putative members of multigene families. They appear to be evolutionarily conserved within citrus species and/or organ- or stress-specifically expressed. Our results represent a starting point regarding the extent of expression pattern differences underlying PRlgf expression and reveal genes that may prove to be useful in studies regarding biotechnological approaches or citrus resistance markers.