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In contrast to desiccation-tolerant 'orthodox' seeds, so-called 'intermediate' seeds cannot survive complete drying and are short-lived. All species of the genus Coffea produce intermediate seeds, but they show a considerable variability in seed desiccation tolerance (DT), which may help to decipher the molecular basis of seed DT in plants. We performed a comparative transcriptome analysis of developing seeds in three coffee species with contrasting desiccation tolerance. Seeds of all species shared a major transcriptional switch during late maturation that governs a general slow-down of metabolism. However, numerous key stress-related genes, including those coding for the late embryogenesis abundant protein EM6 and the osmosensitive calcium channel ERD4, were up-regulated during DT acquisition in the two species with high seed DT, C. arabica and C. eugenioides. By contrast, we detected up-regulation of numerous genes involved in the metabolism, transport, and perception of auxin in C. canephora seeds with low DT. Moreover, species with high DT showed a stronger down-regulation of the mitochondrial machinery dedicated to the tricarboxylic acid cycle and oxidative phosphorylation. Accordingly, respiration measurements during seed dehydration demonstrated that intermediate seeds with the highest DT are better prepared to cease respiration and avoid oxidative stresses.

Antigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show that Ki-67 controls heterochromatin organisation. Altering Ki-67 expression levels did not significantly affect cell proliferation in vivo. Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently. Conversely, upregulation of Ki-67 expression in differentiated tissues did not prevent cell cycle arrest. Ki-67 interactors included proteins involved in nucleolar processes and chromatin regulators. Ki-67 depletion disrupted nucleologenesis but did not inhibit pre-rRNA processing. In contrast, it altered gene expression. Ki-67 silencing also had wide-ranging effects on chromatin organisation, disrupting heterochromatin compaction and long-range genomic interactions. Trimethylation of histone H3K9 and H4K20 was relocalised within the nucleus. Finally, overexpression of human or Xenopus Ki-67 induced ectopic heterochromatin formation. Altogether, our results suggest that Ki-67 expression in proliferating cells spatially organises heterochromatin, thereby controlling gene expression., SCOPUS: ar.j, info:eu-repo/semantics/published

The unicellular pathogenic protozoan Trypanosoma brucei gambiense is responsible for the chronic form of sleeping sickness. This vector-borne disease is transmitted to humans by the tsetse fly of the group Glossina palpalis, including the subspecies G. p. gambiensis, in which the parasite completes its developmental cycle. Sleeping sickness control strategies can therefore target either the human host or the fly vector. Indeed, suppression of one step in the parasite developmental cycle could abolish parasite transmission to humans, with consequences on the spreading of the disease. In order to develop this type of approach, we have identified, at the proteome level, events resulting from the tripartite interaction between the tsetse fly G. p. gambiensis, its microbiome, and the trypanosome. Proteomes were analyzed from four biological replicates of midguts from flies sampled 3 days post-feeding on either a trypanosome-infected (stimulated flies) or a non-infected (non-stimulated flies) bloodmeal. Over 500 proteins were identified in the midguts of flies from both feeding groups, 13 of which were shown to be differentially expressed in trypanosome-stimulated vs. non-stimulated flies. Functional annotation revealed that several of these proteins have important functions that could be involved in modulating the fly infection process by trypanosomes (and thus fly vector competence), including anti-oxidant and anti-apoptotic, cellular detoxifying, trypanosome agglutination, and immune stimulating or depressive effects. The results show a strong potential for diminishing or even disrupting fly vector competence, and their application holds great promise for improving the control of sleeping sickness.

The unicellular pathogenic protozoan Trypanosoma brucei gambiense is responsible for the chronic form of sleeping sickness. This vector-borne disease is transmitted to humans by the tsetse fly of the group Glossina palpalis, including the subspecies G. p. gambiensis, in which the parasite completes its developmental cycle. Sleeping sickness control strategies can therefore target either the human host or the fly vector. Indeed, suppression of one step in the parasite developmental cycle could abolish parasite transmission to humans, with consequences on the spreading of the disease. In order to develop this type of approach, we have identified, at the proteome level, events resulting from the tripartite interaction between the tsetse fly G. p. gambiensis, its microbiome, and the trypanosome. Proteomes were analyzed from four biological replicates of midguts from flies sampled 3 days post-feeding on either a trypanosome-infected (stimulated flies) or a non-infected (non-stimulated flies) bloodmeal. Over 500 proteins were identified in the midguts of flies from both feeding groups, 13 of which were shown to be differentially expressed in trypanosome-stimulated vs. non-stimulated flies. Functional annotation revealed that several of these proteins have important functions that could be involved in modulating the fly infection process by trypanosomes (and thus fly vector competence), including anti-oxidant and anti-apoptotic, cellular detoxifying, trypanosome agglutination, and immune stimulating or depressive effects. The results show a strong potential for diminishing or even disrupting fly vector competence, and their application holds great promise for improving the control of sleeping sickness.

The aim of this study was to assess whether endosperm-specific carotenoid biosynthesis influenced core metabolic processes in maize embryo and endosperm and how global seed metabolism adapted to this expanded biosynthetic capacity. Although enhancement of carotenoid biosynthesis was targeted to the endosperm of maize kernels, a concurrent up-regulation of sterol and fatty acid biosynthesis in the embryo was measured. Targeted terpenoid analysis, and non-targeted metabolomic, proteomic, and transcriptomic profiling revealed changes especially in carbohydrate metabolism in the transgenic line. In-depth analysis of the data, including changes of metabolite pools and increased enzyme and transcript concentrations, gave a first insight into the metabolic variation precipitated by the higher up-stream metabolite demand by the extended biosynthesis capacities for terpenoids and fatty acids. An integrative model is put forward to explain the metabolic regulation for the increased provision of terpenoid and fatty acid precursors, particularly glyceraldehyde 3-phosphate and pyruvate or acetyl-CoA from imported fructose and glucose. The model was supported by higher activities of fructokinase, glucose 6-phosphate isomerase, and fructose 1,6-bisphosphate aldolase indicating a higher flux through the glycolytic pathway. Although pyruvate and acetyl-CoA utilization was higher in the engineered line, pyruvate kinase activity was lower. A sufficient provision of both metabolites may be supported by a by-pass in a reaction sequence involving phosphoenolpyruvate carboxylase, malate dehydrogenase, and malic enzyme.

The study of phosphoproteome on a global scale represents one of the challenges in the post-genomic era. Here, we propose an integrated procedure starting from the crude protein extract, that consists of sequential purification steps, and ending up in the identification of phosphorylation sites. This involves (i) an enrichment in phosphoproteins with a commercially available chromatography matrix, (ii) a 2-D gel analysis of the enriched fraction followed by the selective staining with the phosphospecific fluorescent dye Pro-Q Diamond, (iii) a phosphopeptide capture, from the tryptic lysate of 2-D spots, using IMAC micro-columns. In the end, the identification of the phosphoproteins and their corresponding phosphorylation sites were achieved by MALDI-TOF-TOF spectrometry. The method was applied to contrasting samples prepared from cell suspension cultures of Arabidopsis thaliana and roots of Medicago truncatula. The results obtained, demonstrated the robustness of the combination of two enrichment stages, sequentially at the protein and at the peptide levels, to analyse phosphoproteins in plants., Evaluation Studies, Journal Article, info:eu-repo/semantics/published

The study of phosphoproteome on a global scale represents one of the challenges in the post-genomic era. Here, we propose an integrated procedure starting from the crude protein extract, that consists of sequential purification steps, and ending up in the identification of phosphorylation sites. This involves (i) an enrichment in phosphoproteins with a commercially available chromatography matrix, (ii) a 2-D gel analysis of the enriched fraction followed by the selective staining with the phosphospecific fluorescent dye Pro-Q Diamond, (iii) a phosphopeptide capture, from the tryptic lysate of 2-D spots, using IMAC micro-columns. In the end, the identification of the phosphoproteins and their corresponding phosphorylation sites were achieved by MALDI-TOF-TOF spectrometry. The method was applied to contrasting samples prepared from cell suspension cultures of Arabidopsis thaliana and roots of Medicago truncatula. The results obtained, demonstrated the robustness of the combination of two enrichment stages, sequentially at the protein and at the peptide levels, to analyse phosphoproteins in plants., Evaluation Studies, Journal Article, info:eu-repo/semantics/published