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ANNOTATION AND EXPRESSION OF FDM-LIKE GENES IN SEXUAL AND APOMICTIC H. PERFORATUM ACCESSIONS
Full text linkApomixis is an asexual reproductive strategy that permits to the inheritance of the maternal
genome over generations without genetic recombination events. From a biological point of view,
this modality of asexual reproduction by seed could be the consequence of heterocronic expression
of genes involved in fundamental aspects of sexual reproduction, such as meiosis and spore
selection, leading to the formation of functional un-reduced gametes which retain the ability to form
an embryo eventually bypassing double fertilization. Although this process is well documented in
many flowering plants, and despite its revolutionary potentials in plant breeding strategies, the
genetic control of apomixes is still not properly understood. Recently, a number of independent
studies on sexual model species provided experimental evidences linking single components of
apomixis to the deregulation of genes involved in the RNA-directed DNA methylation pathway
(RdDM). RNA-directed DNA methylation refers to a specific process in which small interfering
RNAs produced by the RNA interference pathway guide de novo methylation of cytosines at the
homologous DNA region, thereby regulating the expression of specific genes. Remarkably, gene
expression studies in the apomictic model species Hypericum perforatum corroborate the
hypothesis that miss expression of genes involved in this pathway is functionally associated with
early events of apomixes in this species. The aim of the present research is a better understanding of
the role of a class of genes involved in the RdDM known as Factor of DNA Methylation (FDM), in
the frame of ovule cell fate change, megagametogenesis and, ultimately, aposporous apomixis. To
this end, computational investigations were performed in order to annotate the H. perforatum FDM
genes. The expression of these genes in H. perforatum pistils and ovules was studied by qPCR and
ISH assays on sexual and apomictic accessions. Furthermore, Arabidopsis thaliana knock out lines
were characterized in order to elucidate the role of specific genes in relation to gametophyte and/or
seed formation. All data will be presented and critically discussed as they prove a better
understanding of molecular bases of apomixes in H. perforatum
Author Correction: Uncovering the sources of DNA found on the Turin Shroud
Full text linkAn amendment to this paper has been published and can be accessed via a link at the top of the paper
ANNOTATION AND EXPRESSION OF FDM-LIKE GENES IN SEXUAL AND APOMICTIC MODEL SPECIES
Full text linkAposporous apomixis is a reproductive strategy that leads to seed production by avoiding
female sporogenesis and, eventually, fertilization. In this peculiar reproductive strategy, embryo and
endosperm are formed from unreduced gametes developing from somatic cells belonging to the
ovule nucellus. Recently gained information on ovule gene expression in the apomictic model
species Hypericum perforatum L. demonstrated that a few genes involved in the RNA-directed
DNA methylation pathway (RdDM) are differentially expressed in ovules collected from apomictic
accessions at pre-meiotic stages. In A. thaliana, the protein Involved in De Novo 2 (IDN2), together
with members of the gene family Factors of DNA Methylation (FDM1, FDM2), acts downstream of
the RdDM. In this pathway, IDN2/FDM complex are recruited to the chromatin by the ra-siRNAPol V transcript duplex, and then bind the un-methylated DNA to promote DNA methylation of
both transposons and protein coding genes. Remarkably, the knock-out of genes involved in the
RdDM in sexually reproducing species such as A. thaliana and Z. mays results in phenotypes
mimicking early events of aposporous apomixis. Taken together, these findings let us to
hypothesize that RNA-directed DNA methylation might be involved in correct patterning of cell
fate determination in the ovule in sexual and apomictic species. This research focuses on genes
belonging to the gene family known as Factors of DNA Methylation (FDM1-5) and their closely
related IDN2 (Involved in De Novo 2). Our research aim is a better understanding of roles played
by these genes in the frame of ovule cell fate determination and gametes formation.
Bioinformatics analyses were performed in order to identify and annotate all gene family
members expressed in H. perforatum ovules. Gene expression differences between pistils collected
from sexual and apomictic accessions were confirmed by qPCR and ISH. Correlated experiments
were performed by taking advantage of mutant lines available for A. thaliana. IDN2 and FDM1-5
knockout lines were analyzed for alterations in total seed set and plant habits. Mutant lines
displayed overlapping phenotypes, including the reduction of seed set. Overall, our phenotypic data
are in line with a sporophytic effect resulting in the ovule abortion in A. thaliana. GUS reporter
lines were adopted to visualize the FDM promoter activity in ovules at different developmental time
points. Furthermore, the development of a protocol suitable for whole-mount qPCR assays allowed
rapid and reliable quantification of gene expression in micro-dissected ovules. Our results elucidate
the role of FDM and IDN2 genes in both sexual and apomicts plants and add new factors affecting
the complex events involved in ovule and gametes formation that should be further investigated
TRANSCRIPTOMIC ANALYSIS OF OVULE-SPECIFIC CELL LINEAGES TO IDENTIFY GENES RELATED TO APOSPOROUS APOMIXIS IN HYPERICUM PERFORATUM L.
Full text linkThe medicinal plant St. John’s wort (Hypericum perforatum L.) is an attractive model system
for the study of aposporous apomixis. The key biological features of apomixis in this species are the
by-passing of meiosis, the differentiation of aposporous initials into embryo sacs containing
unreduced egg cells, their autonomous development in functional embryos without fertilization, and
the formation of viable endosperm either via fertilization-independent means or following
fertilization with a sperm nucleus. The aim of this research is to define gene expression changes
occurring in the nucellar cell types of the ovules primarily involved in the differentiation of the
megaspore mother cells and aposporous initials. To this end, a laser-capture microdissection
approach was adopted in combination with the RNA-seq technology in order to restrict the frame of
our investigations to a specific portion of the ovule, i.e. the nucellus, at developmental stages
preceding the differentiation of aposporous initials. Overall, our gene expression analysis identified
270 and 81 unigenes that were found significantly up- and down-regulated between ovules collected
from sexual and apomictic accessions. Ontological annotation of differentially expressed genes
indicated that genes up-regulated in apomictic ovules were significantly enriched in ontological
terms related to the RNA-directed DNA polymerase activity and the RNA binding. Among these
genes, several actors of the RdDM pathway were found, suggesting that the phenotypic expression
of early events of aposporous apomixis is associated to changes in de novo DNA methylation
mediated by small RNAs. Furthermore, as deregulation of single components of the sexual
developmental pathway is believed to be a trigger of the apomictic reproductive program, genes
involved in sporogenesis, gametogenesis and response to hormonal stimuli were annotated and
investigated in great detail. The expression analysis of candidate genes was performed not only by
Real-Time qPCR but also by ISH assays in order to verify the temporal and spatial expression
patterns of selected transcripts in the ovule. Finally, the activity of specific genes in relation to
embryo sac and/or embryo formation was investigated by using A. thaliana knock-out lines.
Overall, our data suggest that phenotypic expression of aposporous apomixis is concomitant with
the modulation of key genes involved in the sexual reproductive pathway, hormones and other
actors likely playing a crucial role in the RNA-directed DNA methylation pathway
Annotation and Expression of IDN2-like and FDM-like genes in sexual and aposporous hypericum perforatum L. Accessions
Full text linkThe protein IDN2, together with the highly similar interactors FDM1 and FDM2, is required for RNA-directed DNA methylation (RdDM) and siRNA production. Epigenetic regulation of gene expression is required to restrict cell fate determination in A. thaliana ovules. Recently, three transcripts sharing high similarity with the A. thaliana IDN2 and FDM1-2 were found to be differentially expressed in ovules of apomictic Hypericum perforatum L. accessions. To gain further insight into the expression and regulation of these genes in the context of apomixis, we investigated genomic, transcriptional and functional aspects of the gene family in this species. The H. perforatum genome encodes for two IDN2-like and 7 FDM-like genes. Differential and heterochronic expression of FDM4-like genes was found in H. perforatum pistils. The involvement of these genes in reproduction and seed development is consistent with the observed reduction of the seed set and high variability in seed size in A. thaliana IDN2 and FDM-like knockout lines. Differential expression of IDN2-like and FDM-like genes in H. perforatum was predicted to affect the network of potential interactions between these proteins. Furthermore, pistil transcript levels are modulated by cytokinin and auxin but the effect operated by the two hormones depends on the reproductive phenotype
Cloning and Bioinformatic Characterization of genes controlling key steps of the fatty acid biosynthesis and lipid breakdown in seeds of Jatropha curcas L
No full textGoing Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Valutazione dei cambiamenti nelle relazioni insetto-microrganismi su gradiente altitudinale
Full text linkI pascoli alpini sono uno dei paesaggi maggiormente diffusi nell’arco alpino nonostante il progressivo abbandono della pastorizia tradizionale e i cambiamenti climatici costituiscono una seria minaccia per questa tipologia di ambiente. Inoltre, ospitano numerosi organismi che si sono adattati ai metodi di allevamento estensivo: ad esempio è stato appurato come la quantità di biomassa presente nell’ecosistema suolo sia ben maggiore della biomassa presente in superficie, pur includendo piante e bestiame. Molti aspetti degli organismi che abitano il suolo e la sua superficie non sono ancora noti, in particolare le interazioni tra microrganismi e invertebrati all’interno dei pascoli lasciano aperti innumerevoli quesiti. Siamo a conoscenza delle relazioni preda-predatore tra microrganismi ed invertebrati, ma sappiamo anche come molti microrganismi siano simbionti con gli artropodi, ad esempio come parte del loro microbiota intestinale. Questi ultimi microrganismi giocano quindi un ruolo importante anche nella salute stessa degli invertebrati del suolo. Considerando che questo tipo di interazioni sono quasi del tutto ignote, lo scopo dello studio è quello di valutare le relazioni tra microrganismi e la fauna del suolo includendo fattori abiotici quali la temperatura, l’umidità del suolo, il contenuto organico del suolo, ma anche fattori biotici, ai fini di comprendere i fattori determinanti la biodiversità del suolo. In aggiunta, lo studio è svolto su gradiente altitudinale per testare gli effetti dei cambiamenti climatici sulla fauna del suolo e sul loro microbiota intestinale. I taxa selezionati per lo studio sono i seguenti: 1) Nematodi come componenti della microfauna e importanti predatori di microrganismi. 2) Collemboli, membri della mesofauna e importanti organismi detritivori.3) Lombrichi, considerati ingegneri del suolo dal momento che giocano un ruolo importante nella struttura del suolo stesso.4) Coleotteri appartenenti alle famiglie degli stafilinidi (Coleoptera: Staphylinidae) e dei carabidi (Coleoptera: Carabidae), i quali sono importanti predatori di altri invertebrati. Grazie alle tecniche di Next Generation Sequencing come la metataxomica, è oggi possibile individuare intere comunità di microrganismi. Le analisi sono effettuate sulla sequenza del gene 16S rDNA per le regioni V4-V5 per quel che riguarda i Prokaryota e la sequenza del gene Internal Transcribed Spacer 1 per i Fungi. Dal momento che le analisi delle sequenze di DNA sono ancora in fase di svolgimento, al Congresso verranno presentati i risultati preliminari dello studio
EPIGENETIC REGULATION OF ENDOREDUPLICATION PROCESS: A KEY ROLE FOR A CORRECT PEACH FRUIT SIZE AND RIPENING TIMING
Full text linkAmong the several phenotypic traits which characterize the fruit quality, the fruit dimension is
one of the major aspects evaluated in the present worldwide socio-economical context, since it
could be directly related to food and human nutrition supply. From a molecular point of view the
fruit growth and the related fruit size are influenced by different biological processes, including
endoreduplication events in pericarp tissue. In plants, endoreduplication can occur in different cell
types, putatively related with peculiar functions. In fruit crops, endoreduplication have been largely
studied during fruit growth from an ultrastructural and cytologic point of view, while the molecular
and genetic basis of this process are still poorly understood. The genetic control of
endoreduplication during fruit growth is functional to a better understanding of regulatory
mechanisms governing fruit size and ripening timing.
In this research we focused our attention on endoreduplication events occurring in different
peach cvs (Prunus persica L. Batsch) characterized by different size and harvest time, that are
Springcrest (early ripening) and Fantasia (late ripening). In addition, we took advantage of a peach
mutation identified in a free-pollinated population of Fantasia named slow ripening (SR). SR
phenotype is associated to an altered transcriptional regulation of genes involved in mesocarp
identity, and shows a reduction of final fruit size together to the loss of fundamental ripening traits.
In the three cultivars, genes activating endoreduplication (orthologous to Arabidopsis WEE1 and
KRP3) are more expressed in Fantasia in comparison to Springcrest and SR. These transcription
profiles are consistent with flow cytometry data that pointed out an altered endoreduplication level
in Springcrest and SR. In parallel, the involvement of specific epigenetic marks (H3K4me3,
H3K27me3 and H3K9ac) were analyzed at the target gene loci with the goal to link fruit
endoreduplication events to epigenetic control
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