Genetic variations in G-quadruplex forming sequences affect the transcription of human disease-related genes.

Guanine-rich DNA strands can fold into non-canonical four-stranded secondary structures named G-quadruplexes (G4s). G4s folded in proximal promoter regions (PPR) are associated either with positive or negative transcriptional regulation. Given that single nucleotide variants (SNVs) affecting G4 folding (G4-Vars) may alter gene transcription, and that SNVs are associated with the human diseases' onset, we undertook a novel comprehensive study of the G4-Vars genome-wide (G4-variome) to find disease-associated G4-Vars located into PPRs. We developed a bioinformatics strategy to find disease-related SNVs located into PPRs simultaneously overlapping with putative G4-forming sequences (PQSs). We studied five G4-Vars disturbing in vitro the folding and stability of the G4s located into PPRs, which had been formerly associated with sporadic Alzheimer's disease (GRIN2B), a severe familiar coagulopathy (F7), atopic dermatitis (CSF2), myocardial infarction (SIRT1) and deafness (LHFPL5). Results obtained in cultured cells for these five G4-Vars suggest that the changes in the G4s affect the transcription, potentially contributing to the development of the mentioned diseases. Collectively, data reinforce the general idea that G4-Vars may impact on the different susceptibilities to human genetic diseases' onset, and could be novel targets for diagnosis and drug design in precision medicine.

Generation of fruit postharvest gene datasets and a novel motif analysis tool for functional studies: uncovering links between peach fruit heat treatment and cold storage responses.

MAIN CONCLUSION: A survey of developed fruit gene-specific datasets and the implementation of a novel cis-element analysis tool indicate specific transcription factors as novel regulatory actors under HT response and CI protection. Heat treatment (HT) prior to cold storage (CS) has been successfully applied to ameliorate fruit chilling injury (CI) disorders. Molecular studies have identified several HT-driven benefits and putative CI-protective molecules and mechanisms. However, bioinformatic tools and analyses able to integrate fruit-specific information are necessary to begin functional studies and breeding projects. In this work, a HT-responsive gene dataset (HTds) and four fruit expression datasets (FEds), containing gene-specific information from several species and postharvest conditions, were developed and characterized. FEds provided information about HT-responsive genes, not only validating their sensitivity to HT in different systems but also revealing most of them as CS-responsive. A special focus was given to peach heat treatment-sensitive transcriptional regulation by the development of a novel Perl motif analysis software (cisAnalyzer) and a curated plant cis-elements dataset (PASPds). cisAnalyzer is able to assess sequence motifs presence, localization, enrichment and discovery on biological sequences. Its implementation for the enrichment analysis of PASPds motifs on the promoters of HTds genes rendered particular cis-elements that indicate certain transcription factor (TF) families as responsible of fruit HT-sensitive transcription regulation. Phylogenetic and postharvest expression data of these TFs showed a functional diversity of TF families, with members able to fulfil roles under HT, CS and/or both treatments. All integrated datasets and cisAnalyzer tool were deposited in FruitGeneDB (https://www.cefobi-conicet.gov.ar/FruitGeneDB/search1.php), a new available database with a great potential for fruit gene functional studies, including the markers of HT and CS responses whose study will contribute to unravel HT-driven CI-protection and select tolerant cultivars.

NADP-Dependent Malic Enzyme 1 Participates in the Abscisic Acid Response in Arabidopsis thaliana.

Arabidopsis thaliana possesses three cytosolic (NADP-ME1-3) and one plastidic (NADP-ME4) NADP-dependent malic enzymes. NADP-ME2 and -ME4 show constitutive expression, in contrast to NADP-ME1 and -ME3, which are restricted to particular tissues. Here, we show that NADP-ME1 transcript and protein were almost undetectable during normal vegetative growth, but gradually increased and reached levels higher than those of the other isoforms in the latest stages of seed development. Accordingly, in knockout nadp-me1 mature seeds the total NADP-ME activity was significantly lower than in wild type mature seeds. The phenotypic analysis of nadp-me1 plants indicated alterations of seed viability and germination. Besides, the treatment with abscisic acid (ABA), NaCl and mannitol specifically induced the accumulation of NADP-ME1 in seedlings. In line with this, nadp-me1 plants show a weaker response of primary and lateral root length and stomatal opening to the presence of ABA. The results suggest that NADP-ME1 plays a specialized role, linked to ABA signaling during the seed development as well as in the response to water deficit stress.

Cell wall-related genes studies on peach cultivars with differential susceptibility to woolliness: looking for candidates as indicators of chilling tolerance.

KEY MESSAGE: The results obtained indicate that a ß-xylosidase gene may act as good indicator of chilling tolerance and provide new insights into the complex issue of peach fruit woolliness. The storage of peaches at low temperatures for prolonged periods can induce a form of chilling injury (CI) called woolliness, characterized by a lack of juiciness and a mealy texture. As this disorder has been associated with abnormal cell wall dismantling, the levels of 12 transcripts encoding proteins involved in cell wall metabolism were analysed in cultivars with contrasting susceptibility to this disorder selected from five melting flesh peach cultivars. The resistant ('Springlady') and susceptible ('Flordaking') cultivars displayed differences in the level of expression of some of the selected genes during fruit softening and in woolly versus non-woolly fruits. From these genes, the level of expression of PpXyl, which encodes for a putative ß-xylosidase, was the one that presented the highest correlation (negative) with the susceptibility to woolliness. PpXyl expression was also analysed in a cultivar ('Rojo 2') with intermediate susceptibility to woolliness, reinforcing the conclusion about the correlation of PpXyl expression to the presence of woolliness symptom. Moreover, the level of expression of PpXyl correlated to protein level detected by Western blot. Analyses of the promoter region of the PpXyl gene (1637 bp) isolated from the three cultivars showed no differences suggesting that cis-elements from other regions of the genome and/or trans elements could be responsible of the differential PpXyl expression patterns. Overall, the results obtained indicate that PpXyl may act as a good indicator of woolliness tolerance and that the regulation of expression of this gene in different cultivars does not depend on sequences upstream the coding sequence.