Mechanisms of Fruit Ripening: Retrospect and Prospects

This paper aims at giving an overview of the progress made during the last decades on the mechanisms of fruit ripening and to present the most recent trends and prospects for the future. Important steps forward will be presented (respiratory climacteric, ethylene biosynthesis and action, isolation of genes involved in the ripening process, biotechnological control of fruit ripening....) by showing how the judicious exploitation of the data published previously, the strategies, methodologies and plant material adopted have been crucial for the advancement of knowledge. Opportunities of co-operation between geneticists and post-harvest physiologists as well as new possibilities offered by genomics, proteomics and metabolomics for the understanding of the fruit ripening process and the development of sensory quality will be developed

Influence of drought and heat stresses in fodder legumes productivity and nutritive value

Plants are often subjected to several environmental constraints in the field, often simultaneously, which significantly affect their productivity. Among these constraints, water deficit and heat stress are the main constraints limiting plant yield in the arid regions of the Mediterranean basin. Both of these abiotic factors are exacerbated by climate change. Fodder legumes provide a rich resource of plant nutrition to human diets, and are vital for food security and sustainable cropping. Numerous biochemical, molecular, and physiological responses are evoked by drought and heat stresses, influencing the fodder crop yield and nutritional value. When plants are water stressed, they are unable to maintain good growth and produce high yields. Drought also reduces the protein content of the plants, which can lead to a decrease in forage quality. Heat can negatively affect the productivity of forage legumes. High temperatures can cause heat stress which can lead to decreased photosynthesis and reduced productivity. In addition, high temperatures can result in decreased forage quality and increased nutrient losses. Exposure to combined water stress and heat can reduce the protein, fiber and mineral content of forage legumes and can also reduce their digestibility. This review summarizes the current status of the effects of drought and heat stresses on forage crops productivity and fodder quality

Sl-ERF2, a Tomato Ethylene Response Factor Involved in Ethylene Response and Seed Germination

Ethylene response factors (ERFs) are plant transcriptional regulators mediating ethylene-dependent gene expression via binding to the GCC motif found in the promoter region of ethylene-regulated genes. We report here on the structural and functional characterization of the tomato Sl-ERF2 gene that belongs to a distinct class of the large ERF gene family. Both spliced and unspliced versions of Sl-ERF2 transcripts were amplified from RNA samples and the search in the public tomato expressed sequence tag (EST) database confirmed the existence of the two transcript species in a number of cDNA libraries. The unspliced transcript contains two open reading frames yielding two hypothetical proteins, a small highly truncated version lacking the APETALA2 domain and a bigger protein lacking the N-terminal MCGGAAII/L consensus peptide specific to ERF members from subfamily IV. Nevertheless, functional Sl-ERF2 protein may only derive from spliced transcripts since, depending on the tissue, the level of the spliced transcript is much higher than that of the unspliced transcript. Sl-ERF2 is expressed in all plant tissues tested, though its transcript accumulates preferentially in germinating seeds and ripening fruit. Overexpression of the Sl-ERF2 gene in transgenic tomato lines results in premature seed germination and enhanced hook formation of darkgrown seedlings, which is indicative of increased ethylene sensitivity. The expression of the mannanase2 gene is upregulated in Sl-ERF2-overexpressing seeds, suggesting that Sl-ERF2 stimulates seed germination through the induction of the mannanase2 gene. It is noteworthy that the exaggerated hook phenotype is abolished when ethylene perception is blocked, strongly suggesting that Sl-ERF2 requires other ethylene-dependent components to impact the hook formation process

Molecular and biochemical characterization of LeCRK1, a ripening-associated tomato CDPK-related kinase

A cDNA clone (LeCRK1), encoding a novel isoform of calcium-dependent protein kinase (CDPK), was isolated by screening a tomato (Lycopersicon esculentum) cDNA library. The protein derived from the full-length sequence indicated that it belongs to the family of CDPK-related kinases (CRKs) and the predicted amino acid sequence shows a modular organization of the protein consisting of different characteristic domains. The kinase domain of LeCRK1 shares a high degree of similarity with the catalytic domain of CDPKs. In contrast to canonical members of the family, LeCRK1 has a degenerate sequence in the C-terminal calmodulinlike domain. LeCRK1 protein was shown to be a functional kinase, but, consistent with the lack of calciumbinding activity, its autophosphorylation activity did not require calcium. LeCRK1 harbours an amphiphilic amino acid region revealed to be a functional calmodulinbinding site by in vitro assay. A putative myristoylation/ palmitoylation sequence has been identified at the N-terminus. Expressing an LeCRK1::GFP fusion protein in the protoplast resulted in its targeting to the plasma membrane. Site-directed mutagenesis of critical amino acids of the myristoylation/palmitoylation consensus sites led to the accumulation of the mutated protein in the cytoplasm, suggesting that the native protein is anchored to the plasma membrane by acylated residues. Expression studies revealed significant accumulation of LeCRK1 transcripts during fruit ripening, although transcripts were also detected in stem, leaf, and flower. LeCRK1 mRNA level in leaves was slightly induced by ethylene and salicylic acid, and upon mechanical wounding and cold treatment. It is noteworthy that LeCRK1 mRNAs were undetectable in different tomato-ripening natural mutants such as NR, Rin, and Nor, suggesting a role in the ripening process

Genetic landscape of 6089 inherited retinal dystrophies affected cases in Spain and their therapeutic and extended epidemiological implications

Inherited retinal diseases (IRDs), defined by dysfunction or progressive loss of photoreceptors, are disorders characterized by elevated heterogeneity, both at the clinical and genetic levels. Our main goal was to address the genetic landscape of IRD in the largest cohort of Spanish patients reported to date. A retrospective hospital-based cross-sectional study was carried out on 6089 IRD affected individuals (from 4403 unrelated families), referred for genetic testing from all the Spanish autonomous communities. Clinical, demographic and familiar data were collected from each patient, including family pedigree, age of appearance of visual symptoms, presence of any systemic findings and geographical origin. Genetic studies were performed to the 3951 families with available DNA using different molecular techniques. Overall, 53.2% (2100/3951) of the studied families were genetically characterized, and 1549 different likely causative variants in 142 genes were identified. The most common phenotype encountered is retinitis pigmentosa (RP) (55.6% of families, 2447/4403). The most recurrently mutated genes were PRPH2, ABCA4 and RS1 in autosomal dominant (AD), autosomal recessive (AR) and X-linked (XL) NON-RP cases, respectively; RHO, USH2A and RPGR in AD, AR and XL for non-syndromic RP; and USH2A and MYO7A in syndromic IRD. Pathogenic variants c.3386G > T (p.Arg1129Leu) in ABCA4 and c.2276G > T (p.Cys759Phe) in USH2A were the most frequent variants identified. Our study provides the general landscape for IRD in Spain, reporting the largest cohort ever presented. Our results have important implications for genetic diagnosis, counselling and new therapeutic strategies to both the Spanish population and other related populations.This work was supported by the Instituto de Salud Carlos III (ISCIII) of the Spanish Ministry of Health (FIS; PI16/00425 and PI19/00321), Centro de Investigación Biomédica en Red Enfermedades Raras (CIBERER, 06/07/0036), IIS-FJD BioBank (PT13/0010/0012), Comunidad de Madrid (CAM, RAREGenomics Project, B2017/BMD-3721), European Regional Development Fund (FEDER), the Organización Nacional de Ciegos Españoles (ONCE), Fundación Ramón Areces, Fundación Conchita Rábago and the University Chair UAM-IIS-FJD of Genomic Medicine. Irene Perea-Romero is supported by a PhD fellowship from the predoctoral Program from ISCIII (FI17/00192). Ionut F. Iancu is supported by a grant from the Comunidad de Madrid (CAM, PEJ-2017-AI/BMD7256). Marta del Pozo-Valero is supported by a PhD grant from the Fundación Conchita Rábago. Berta Almoguera is supported by a Juan Rodes program from ISCIII (JR17/00020). Pablo Minguez is supported by a Miguel Servet program from ISCIII (CP16/00116). Marta Corton is supported by a Miguel Servet program from ISCIII (CPII17/00006). The funders played no role in study design, data collection, data analysis, manuscript preparation and/or publication decisions

CIBERER : Spanish national network for research on rare diseases: A highly productive collaborative initiative

Altres ajuts: Instituto de Salud Carlos III (ISCIII); Ministerio de Ciencia e Innovación.CIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research

Expression of Structural Flavonoid Biosynthesis Genes in Dark-Blue and White Myrtle Berries (<i>Myrtus communis</i> L.)

Within the myrtle (Myrtus communis L.) species, different genotypes may produce dark-blue berries or white berries depending on the peel color upon ripening. One dark-blue cultivar and one white myrtle cultivar were used to study the molecular mechanisms underlying flavonoid biosynthesis. The relative expression levels of common (PAL, CHS, CHI, DFR and LDOX) and specific (FLS, ANR, LAR and UFGT) flavonoid genes were analyzed during fruit development by means of quantitative real-time polymerase chain reaction (RT-qPCR). Moreover, the anthocyanin content was determined, and it showed an increase with the ripening of the berries of the dark-blue cultivar. The results showed an increased transcript abundance of PAL, CHI, DFR, LDOX and UFGT gene expression in the dark-blue cultivar compared to the white one, as well as a strong positive correlation between the changes in gene expression and anthocyanin accumulation. The transcript levels of UFGT showed sharp increases at 150 and 180 days after full blooming (DAF) in the dark-blue cultivar, which corresponded with anthocyanin accumulation. However, ripening seemed to modulate the expression of genes implicated in flavonols (i.e., FLS) and flavan-3-ols (i.e., LAR and ANR) in different manners. However, whereas FLS transcript accumulation increased at the end of the ripening period in the dark-blue cultivar, LAR and ANR gene expression decreased in both cultivars

Expression of Structural Flavonoid Biosynthesis Genes in Dark-Blue and White Myrtle Berries (Myrtus communis L.)

Within the myrtle (Myrtus communis L.) species, different genotypes may produce dark-blue berries or white berries depending on the peel color upon ripening. One dark-blue cultivar and one white myrtle cultivar were used to study the molecular mechanisms underlying flavonoid biosynthesis. The relative expression levels of common (PAL, CHS, CHI, DFR and LDOX) and specific (FLS, ANR, LAR and UFGT) flavonoid genes were analyzed during fruit development by means of quantitative real-time polymerase chain reaction (RT-qPCR). Moreover, the anthocyanin content was determined, and it showed an increase with the ripening of the berries of the dark-blue cultivar. The results showed an increased transcript abundance of PAL, CHI, DFR, LDOX and UFGT gene expression in the dark-blue cultivar compared to the white one, as well as a strong positive correlation between the changes in gene expression and anthocyanin accumulation. The transcript levels of UFGT showed sharp increases at 150 and 180 days after full blooming (DAF) in the dark-blue cultivar, which corresponded with anthocyanin accumulation. However, ripening seemed to modulate the expression of genes implicated in flavonols (i.e., FLS) and flavan-3-ols (i.e., LAR and ANR) in different manners. However, whereas FLS transcript accumulation increased at the end of the ripening period in the dark-blue cultivar, LAR and ANR gene expression decreased in both cultivars

Expression profiles and DNA-binding affinity of five ERF genes in bunches of Vitis vinifera cv. Cardinal treated with high levels of CO2 at low temperature

Ethylene response factors (ERFs) play an important role in plants by regulating defense response through interaction with various stress pathways. After harvest, table grapes (Vitis vinifera L.) are subject to a range of problems associated with postharvest storage at 0ºC, such as fungal attack, water loss and rachis browning. The application of a 3-day high CO2 treatment maintained fruit quality and activated the induction of transcription factors belonging to different families such as ERF. In this paper, we have isolated five VviERFs from table grapes cv. Cardinal, whose deduced amino acid sequence contained the conserved apetalous (AP2)/ERF domain. The phylogeny and putative conserved motifs in VviERFs were analyzed and compared with those previously reported in Vitis. VviERFs-c gene expression was studied by quantitative real-time RT-PCR (RT-qPCR) in the different tissues of bunches stored at low temperature and treated with high levels of CO2. The results showed that in most of the tissues analyzed, VviERFs-c gene expression was induced by the storage under normal atmosphere although the application of high levels of CO2 caused a greater increase in the VviERFs-c transcript accumulation. The promoter regions of two PRs (Pathogenesis Related Proteins), Vcchit1b and Vcgns1, were obtained and the in silico analysis revealed the presence of a cis-acting ethylene response element (GCC box). In addition, gene expression of these two PRs was analyzed in the pulp and rachis of CO2-treated and non-treated table grapes stored at 0ºC and results showed significant correlations with VviERF2-c and VviERF6L7-c gene expression in rachis, and between VviERF11-c and Vcchit1b in pulp. Finally by using electro mobility shift assays (EMSA), we denoted differences in binding of VviERFs to the GCC sequences present in the promoters of both PRs, with VviERF6L7-c being the only member which did not bind to any tested probe. Overall, our results suggest that the beneficial effect of high CO2 treatment maintaining table grape quality seems to be mediated by the regulation of ERFs and in particular VviERF2-c might play an important role by modulating the expression of PRs