Impaired myocardial metabolic reserve and substrate selection flexibility during stress in patients with idiopathic dilated cardiomyopathy

Under resting conditions, the failing heart shifts fuel use toward greater glucose and lower free fatty acid (FFA) oxidation. We hypothesized that chronic metabolic abnormalities in patients with dilated cardiomyopathy (DCM) are associated with the absence of the normal increase in myocardial glucose uptake and maintenance of cardiac mechanical efficiency in response to pacing stress. In 10 DCM patients and 6 control subjects, we measured coronary flow by intravascular ultrasonometry and sampled arterial and coronary sinus blood. Myocardial metabolism was determined at baseline, during atrial pacing at 130 beats/min, and at 15 min of recovery by infusion of [(3)H]oleate and [(13)C]lactate and measurement of transmyocardial arteriovenous differences of oxygen and metabolites. At baseline, DCM patients showed depressed coronary flow, reduced uptake and oxidation of FFA, and preferential utilization of carbohydrates. During pacing, glucose uptake increased by 106% in control subjects but did not change from baseline in DCM patients. Lactate release increased by 122% in DCM patients but not in control subjects. Cardiac mechanical efficiency in DCM patients was not different compared with control subjects at baseline but was 34% lower during stress. Fatty acid uptake and oxidation did not change with pacing in either group. Our results show that in DCM there is preferential utilization of carbohydrates, which is associated with reduced flow and oxygen consumption at rest and an impaired ability to increase glucose uptake during stress. These metabolic abnormalities might contribute to progressive cardiac deterioration and represent a target for therapeutic strategies aimed at modulating cardiac substrate utilization.     

Nucleotide Diversity of the Coding and Promoter Regions of <Emphasis Type="Italic">DREB1D</Emphasis>, a Candidate Gene for Drought Tolerance in <Emphasis Type="Italic">Coffea</Emphasis> 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.     

Lipid transfer proteins in coffee: isolation of Coffea orthologs,Coffea arabica homeologs,expression during coffee fruit development and promoter analysis in transgenic tobacco plants

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 β-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.     

CRISPR/Cas9-mediated efficient targeted mutagenesis has the potential to accelerate the domestication of <Emphasis Type="Italic">Coffea canephora</Emphasis>

Genome editing, which is an unprecedented technological breakthrough, has provided a valuable means of creating targeted mutations in plant genomes. In this study, we developed a genomic web tool to identify all gRNA target sequences in the coffee genome, along with potential off-targets. In all, 8,145,748 CRISPR guides were identified in the draft genome of Coffea canephora corresponding to 5,338,568 different sequences and, of these, 4,655,458 were single, and 514,591 were covering exons. The proof of concept was established by targeting the phytoene desaturase gene (CcPDS) using the Agrobacterium tumefaciens transformation technique and somatic embryogenesis as the plant regeneration method. An analysis of the RNA-guided genome-editing events showed that 22.8% of the regenerated plants were heterozygous mutants and 7.6% were homozygous mutants. Mutation efficiency at the target site was estimated to be 30.4%. We demonstrated that genome editing by the CRISPR/Cas9 method is an efficient and reliable way of knocking out genes of agronomic interest in the coffee tree, opening up the way for coffee molecular breeding. Our results also showed that the use of somatic embryogenesis, as the method for regenerating genome-edited plants, could restrict the choice of targeted genes to those that are not essential to the embryo development and germination steps.     

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 conjugative plasmid vector for promoter analysis in several cyanobacteria of the genera Synechococcus and Synechocystis

A promoter-probe vector, pSB2A, based on the plasmid RSF1010 and the promoterless chloramphenicol acetyl transferase (cat) reporter gene, has been constructed. pSB2A appeared to be most efficiently transferred by conjugation to the widely used cyanobacteria Synechocystis strains PCC6803 (S.6803) and PCC6714 (S.6714) and Synechococcus strains PCC7942 (S.7942) and PCC6301 (S.6301), where it replicates stably even though it contains no cyanobacterial DNA. Using pSB2A we found that (1) a light-regulated promoter from S.6803 remains controlled by light intensity in S.7942 while it is silent in Escherichia coli, and (2) the E. coli tac promoter behaves as a strong and light-independent promoter in the four cyanobacterial hosts tested.Service de Biochimie et Génétique Moléculaire