Gene expression analysis of Coffea arabica seeds processed under different post-harvest processing methods

The mode of coffee processing, either the wet or dry method, determines the characteristic flavour and establishes the differences in quality of the final green coffee produced. The present study focused mainly on identifying the differential gene expression in green coffee seeds of Brazilian arabica coffee (Coffea arabica L.) among samples prepared under three different post-harvest treatments (natural, washed and semi washed method) and grown in two different locations. Expression levels of 16 genes of interest were measured. These genes are involved in various cellular, metabolic and biochemical activities influencing levels of certain compounds, such as lipids, carbohydrates, caffeine and chlorogenic acid, associated with quality characteristics of the beverage. Microarray experiments were designed with cDNA probe sequences. Microarray data was analyzed to identify the differences in gene expression between two altitudes and between two variables: location and post-harvest treatment. Cluster analysis was carried out with samples showing similar patterns, which are characteristic to the group. With this approach, it was possible to identify the important genes in C. arabica seeds that have differential (increased or decreased) expression levels. It was also seen that between the location and treatments, location profoundly impacts the levels of gene expression in samples

A single polyploidization event at the origin of the tetraploid genome of Coffea arabica is responsible for the extremely low genetic variation in wild and cultivated germplasm

The genome of the allotetraploid species Coffea arabica L. was sequenced to assemble independently the two component subgenomes (putatively deriving from C. canephora and C. eugenioides) and to perform a genome-wide analysis of the genetic diversity in cultivated coffee germplasm and in wild populations growing in the center of origin of the species. We assembled a total length of 1.536 Gbp, 444 Mb and 527 Mb of which were assigned to the canephora and eugenioides subgenomes, respectively, and predicted 46,562 gene models, 21,254 and 22,888 of which were assigned to the canephora and to the eugeniodes subgenome, respectively. Through a genome-wide SNP genotyping of 736 C. arabica accessions, we analyzed the genetic diversity in the species and its relationship with geographic distribution and historical records. We observed a weak population structure due to low-frequency derived alleles and highly negative values of Taijma's D, suggesting a recent and severe bottleneck, most likely resulting from a single event of polyploidization, not only for the cultivated germplasm but also for the entire species. This conclusion is strongly supported by forward simulations of mutation accumulation. However, PCA revealed a cline of genetic diversity reflecting a west-to-east geographical distribution from the center of origin in East Africa to the Arabian Peninsula. The extremely low levels of variation observed in the species, as a consequence of the polyploidization event, make the exploitation of diversity within the species for breeding purposes less interesting than in most crop species and stress the need for introgression of new variability from the diploid progenitors

Caratterizzazione di geni di coffea arabica L.correlati alle caratteristiche di qualità in tazza della bevanda di caffè.

2006/2007La bevanda di caffè è bevuta ed apprezzata in tutto il mondo per le sue qualità organolettiche. Queste sono numerose e sfaccettate in molti diversi aspetti, che vanno dal contenuto in caffeina alle proprietà antiossidanti. Inoltre, il caffè presenta un ricchissimo profilo aromatico, composto da circa un migliaio di composti volatili che contribuiscono a dare alla bevanda il suo aroma caratteristico. Questo complesso aroma è fortemente influenzato da numerosi fattori quali l’origine geografica della pianta, la sua varietà botanica e le modalità di lavorazione del frutto e del seme dopo la raccolta. Tutti questi fattori contribuiscono a dare a ciascun tipo di caffè un aroma unico, discriminabile sia dal punto di vista sensoriale che chimico. Le sostanze che compongono il profilo aromatico della bevanda di caffè sono state studiate sotto molti aspetti, da quello analitico a quello sensoriale. Non è invece stato svolto alcuno studio sull’origine biochimica e biomolecolare di tali composti nella pianta di Coffea arabica, nonostante ricerche in tal senso siano già state svolte in altre piante importanti dal punto di vista alimentare ed economico. Il presente lavoro si è posto l’obiettivo di identificare e caratterizzare alcuni geni della pianta di Coffea arabica, che potessero essere correlati alla qualità percepita dal consumatore finale nella tazzina di caffè. I geni che meglio soddisfacevano questi requisiti sono stati individuati nelle monoterpene sintasi. I monoterpeni sono infatti tra i più importanti composti odorosi vegetali, responsabili delle note aromatiche positive di tè, caffè, vino, presenti negli olii essenziali di numerose piante aromatiche quali salvia, basilico, menta, e nelle resine di pini ed abeti. Sono stati quindi disegnati dei primer degenerati sulla base delle sequenze di monoterpene sintasi presenti nei database pubblici. Questi primer hanno consentito di amplificare 3 trascritti di Coffea arabica, la cui sequenza completa è poi stata ottenuta mediante la tecnica RACE. Queste putative monoterpene sintasi sono state isolate da vari cDNA sintetizzati da fiore, drupa a vari stadi di maturazione, e seme. L’analisi di queste sequenze mediante BLAST e analisi filogenetica ha permesso di stabilire la loro elevata omologia con geni noti di monoterpene sintasi, confermando quindi il loro status di putative monoterpene sintasi. Questi trascritti, denominati CaMTS (Coffea arabica MonoTerpene Sintasi) sono i primi geni isolati dalla pianta di caffè a poter essere correlati alla qualità percepita in tazza in termini di aroma, percezione olfattiva e gustativa della bevanda, e quindi -in ultima analisi- di apprezzamento edonistico del prodotto. La loro importanza è quindi notevole, anche se la complessità della composizione chimica del caffè suggerisce l’esistenza di una famiglia genica molto numerosa, di cui i 3 geni individuati sono sicuramente solo una piccola parte. Come evidenziato dalle analisi filogenetiche, però, i geni di monoterpene sintasi tendono ad essere molto simili all’interno di una stessa specie, per cui la conoscenza dei primi enzimi di questa famiglia in Coffea arabica sarà presupposto fondamentale per la caratterizzazione di ulteriori geni di questo tipo, fatto che porterà gradualmente ad approfondire la conoscenza dei meccanismi che legano la genetica della pianta di caffè alla qualità del prodotto finale.XX Ciclo197

Aquaporins in Coffea arabica L.: Identification, expression, and impacts on plant water relations and hydraulics

Plant aquaporins (AQPs) are involved in the transport of water and other small solutes across cell membranes, and thus play major roles in the regulation of plant water balance, as well as in growth regulation and response to abiotic stress factors. Limited information is currently available about the presence and role of AQPs in Coffea arabica L., despite the economic importance of the species and its vulnerability to drought stress. We identified candidate AQP genes by screening a proprietary C. arabica transcriptome database, resulting in the identification of nine putative aquaporins. A phylogenetic analysis based on previously characterized AQPs from Arabidopsis thaliana and Solanum tuberosum allowed to assign the putative coffee AQP sequences to the Tonoplast (TIP) and Plasma membrane (PIP) subfamilies. The possible functional role of coffee AQPs was explored by measuring hydraulic conductance and aquaporin gene expression on leaf and root tissues of two-year-old plants (. C. arabica cv. Pacamara) subjected to different experimental conditions. In a first experiment, we tested plants for root and leaf hydraulic conductance both before dawn and at mid-day, to check the eventual impact of light on AQP activity and plant hydraulics. In a second experiment, we measured plant hydraulic responses to different water stress levels as eventually affected by changes in AQPs expression levels. Our results shed light on the possible roles of AQPs in the regulation of C. arabica hydraulics and water balance, opening promising research lines to improve the sustainability of coffee cultivation under global climate change scenarios

Lipase activity and antioxidant capacity in coffee (Coffea arabica L.) seeds during germination

In this paper, lipase activity was characterized in coffee (Coffea arabica L.) seeds to determine its involvement in lipid degradation during germination. The lipase activity, evaluated by a colorimetric method, was already present before imbibition of seeds and was further induced during the germination process. The activity showed a biphasic behaviour, which was similar in seeds either with or without endocarp (parchment), even though the phenomenon showed a delay in the former. The enzymatic activity was inhibited by tetrahydrolipstatin (THL), a selective and irreversible inhibitor of lipases, and by a polyclonal antibody raised against purified alkaline lipase from castor bean. The immunochemical analysis evidenced a protein of ca. 60 kDa, cross-reacting with an anti-lipase antibody, in coffee samples obtained from seeds of both types. Gas chromatographic analyses of free fatty acid (FFA) content confirmed the differences shown in the lipolytic activity of the samples with or without parchment, since FFA levels increased more rapidly in samples without parchment. Finally, the analyses of the antioxidant capacity showed that the presence of parchment was crucial for lowering the oxidation of the lipophylic fraction, being the seeds with parchment less prone to oxidation processes

Rice Husk as an Inexpensive Renewable Immobilization Carrier for Biocatalysts Employed in the Food, Cosmetic and Polymer Sectors

The high cost and environmental impact of fossil-based organic carriers represent a critical bottleneck to their use in large-scale industrial processes. The present study demonstrates the applicability of rice husk as inexpensive renewable carrier for the immobilization of enzymes applicable sectors where the covalent anchorage of the protein is a pre-requisite for preventing protein contamination while assuring the recyclability. Rice husk was oxidized and then functionalized with a di-amino spacer. The morphological characterization shed light on the properties that affect the functionalization processes. Lipase B from Candida antarctica (CaLB) and two commercial asparaginases were immobilized covalently achieving higher immobilization yield than previously reported. All enzymes were immobilized also on commercial epoxy methacrylic resins and the CaLB immobilized on rice husk demonstrated a higher efficiency in the solvent-free polycondensation of dimethylitaconate. CaLB on rice husk appears particularly suitable for applications in highly viscous processes because of the unusual combination of its low density and remarkable mechanical robustness. In the case of the two asparaginases, the biocatalyst immobilized on rice husk performed in aqueous solution at least as efficiently as the enzyme immobilized on methacrylic resins, although the rice husk loaded a lower amount of protein

A chromosome-scale assembly reveals chromosomal aberrations and exchanges generating genetic diversity in Coffea arabica germplasm

Abstract In order to better understand the mechanisms generating genetic diversity in the recent allotetraploid species Coffea arabica, here we present a chromosome-level assembly obtained with long read technology. Two genomic compartments with different structural and functional properties are identified in the two homoeologous genomes. The resequencing data from a large set of accessions reveals low intraspecific diversity in the center of origin of the species. Across a limited number of genomic regions, diversity increases in some cultivated genotypes to levels similar to those observed within one of the progenitor species, Coffea canephora, presumably as a consequence of introgressions deriving from the so-called Timor hybrid. It also reveals that, in addition to few, early-occurring exchanges between homoeologous chromosomes, there are numerous recent chromosomal aberrations including aneuploidies, deletions, duplications and exchanges. These events are still polymorphic in the germplasm and could represent a fundamental source of genetic variation in such a lowly variable species

Il danno alla persona da compromissione della capacità lavorativa. Documento elaborato dalla Commissione di Studio costituita dalla Federazione delle Associazioni Medico-Legali Italiane - F.A.M.L.I.

In questo volume sono raccolti i lavori della Commissione di studio istituita dalla Federazione delle Associazioni Medico-Legali Italiane (F.A.M.L.I.) per elaborare una criteriologia di valutazione della compromissione della capacità lavorativa non soltanto largamente condivisa nel contesto operativo medico-legale, ma anche coerente con assetto giuridico del danno alla persona, innovato dalle note sentenze emesse nel 2003 della Corte Costituzionale e di Cassazione, nonché aderente ai più recenti orientamenti di quest'ultima in materia di prova e risarcibilità del danno patrimoniale e alle correnti caratteristiche del "mercato del lavoro"

Dominant negative variants in IKZF2 cause ICHAD syndrome, a new disorder characterised by immunodysregulation, craniofacial anomalies, hearing impairment, athelia and developmental delay

BackgroundHelios (encoded by IKZF2), a member of the Ikaros family of transcription factors, is a zinc finger protein involved in embryogenesis and immune function. Although predominantly recognised for its role in the development and function of T lymphocytes, particularly the CD4+ regulatory T cells (Tregs), the expression and function of Helios extends beyond the immune system. During embryogenesis, Helios is expressed in a wide range of tissues, making genetic variants that disrupt the function of Helios strong candidates for causing widespread immune-related and developmental abnormalities in humans.MethodsWe performed detailed phenotypic, genomic and functional investigations on two unrelated individuals with a phenotype of immune dysregulation combined with syndromic features including craniofacial differences, sensorineural hearing loss and congenital abnormalities.ResultsGenome sequencing revealed de novo heterozygous variants that alter the critical DNA-binding zinc fingers (ZFs) of Helios. Proband 1 had a tandem duplication of ZFs 2 and 3 in the DNA-binding domain of Helios (p.Gly136_Ser191dup) and Proband 2 had a missense variant impacting one of the key residues for specific base recognition and DNA interaction in ZF2 of Helios (p.Gly153Arg). Functional studies confirmed that both these variant proteins are expressed and that they interfere with the ability of the wild-type Helios protein to perform its canonical function—repressing IL2 transcription activity—in a dominant negative manner.ConclusionThis study is the first to describe dominant negative IKZF2 variants. These variants cause a novel genetic syndrome characterised by immunodysregulation, craniofacial anomalies, hearing impairment, athelia and developmental delay

De novo missense variants in phosphatidylinositol kinase PIP5KIγ underlie a neurodevelopmental syndrome associated with altered phosphoinositide signaling

Phosphoinositides (PIs) are membrane phospholipids produced through the local activity of PI kinases and phosphatases that selectively add or remove phosphate groups from the inositol head group. PIs control membrane composition and play key roles in many cellular processes including actin dynamics, endosomal trafficking, autophagy, and nuclear functions. Mutations in phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] phosphatases cause a broad spectrum of neurodevelopmental disorders such as Lowe and Joubert syndromes and congenital muscular dystrophy with cataracts and intellectual disability, which are thus associated with increased levels of PI(4,5)P2. Here, we describe a neurodevelopmental disorder associated with an increase in the production of PI(4,5)P2 and with PI-signaling dysfunction. We identified three de novo heterozygous missense variants in PIP5K1C, which encodes an isoform of the phosphatidylinositol 4-phosphate 5-kinase (PIP5KIγ), in nine unrelated children exhibiting intellectual disability, developmental delay, acquired microcephaly, seizures, visual abnormalities, and dysmorphic features. We provide evidence that the PIP5K1C variants result in an increase of the endosomal PI(4,5)P2 pool, giving rise to ectopic recruitment of filamentous actin at early endosomes (EEs) that in turn causes dysfunction in EE trafficking. In addition, we generated an in vivo zebrafish model that recapitulates the disorder we describe with developmental defects affecting the forebrain, including the eyes, as well as craniofacial abnormalities, further demonstrating the pathogenic effect of the PIP5K1C variants. We describe a neurodevelopmental disorder associated with de novo gain-of-function variants in PIP5KIγ kinase. The variants cause perturbed endosomal function resulting from increased production of phosphatidylinositol 4,5 bisphosphate and enhanced association of F-actin at endosomes. Moreover, mutant zebrafish larvae recapitulate the phenotypes observed in affected individuals from our cohort