Portuguese wild grapevine genome re-sequencing (Vitis vinifera sylvestris)

Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-76012-6.The first genome of Vitis vinifera vinifera (PN40024), published in 2007, boosted grapevine related studies. While this reference genome is a suitable tool for the overall studies in the field, it lacks the ability to unveil changes accumulated during V. v. vinifera domestication. The subspecies V. v. sylvestris preserves wild characteristics, making it a good material to provide insights into V. v. vinifera domestication. The difference in the reproductive strategy between both subspecies is one of the characteristics that set them apart. While V. v. vinifera flowers are hermaphrodite, V. v. sylvestris is mostly dioecious. In this paper, we compare the re-sequencing of the genomes from a male and a female individual of the wild sylvestris, against the reference vinifera genome (PN40024). Variant analysis reveals a low number but with high impact modifications in coding regions, essentially non-synonymous single nucleotide polymorphisms and frame shifts caused by insertions and deletions. The sex-locus was manually inspected, and the results obtained are in line with the most recent works related with wild grapevine sex. In this paper we also describe for the first time RNA editing in transcripts of 14 genes in the sex-determining region, including VviYABBY and VviPLATZ.Tis work was supported by Fundação para a Ciência e Tecnologia (FCT) through the Research Center LEAF (UIDP/04129/2020). Authors JLCoito, MJNRamos, MRocheta, were funded by FCT fellowships SFRH/ BD/85824/2012 and CEECIND2017, SFRH/BD/110274/2015, SFRH/BPD/64905/2009, respectively

RNA editing in inflorescences of wild grapevine unveils association to sex and development

RNA editing challenges the central dogma of molecular biology, by modifying the genetic information at the transcription level. Recent reports, suggesting increased levels of RNA editing in plants, raised questions on the nature and dynamics of such events during development. We here report the occurrence of distinct RNA editing patterns in wild Vitis flowers during development, with twelve possible RNA editing modifications observed for the first time in plants. RNA editing events are gender and developmental stage specific, identical in subsequent years of this perennial species and with distinct nucleotide frequencies neighboring editing sites on the 5' and 3' flanks. The transcriptome dynamics unveils a new regulatory layer responsible for gender plasticity enhancement or underling dioecy evolution in Vitis

Developmental Regulation of Transcription in Touriga Nacional Berries under Deficit Irrigation

Grapevine (Vitis vinifera L.) is one of the most economically important crops worldwide, especially due to the economic relevance of wine production. Abiotic stress, such as drought, may contribute to low yield, shifts in quality, and important economic loss. The predicted climate change phenomena point to warmer and dryer Mediterranean environmental conditions; as such, it is paramount to study the effects of abiotic stress on grapevine performance. Deficit irrigation systems are applied to optimize water use efficiency without compromising berry quality. In this research, the effect of two deficit irrigation strategies, sustained deficit irrigation (SDI) and regulated deficit irrigation (RDI), in the grape berry were assessed. The effects of different levels of drought were monitored in Touriga Nacional at key stages of berry development (pea size, véraison, and full maturation) through RNA-Seq transcriptome analysis and by specific differentially expressed genes (DEGs) monitoring through RT-qPCR. Handy datasets were obtained by bioinformatics analysis of raw RNA-Seq results. The dominant proportion of transcripts was mostly regulated by development, with véraison showing more upregulated transcripts. Results showed that primary metabolism is the functional category more severely affected under water stress. Almost all DEGs selected for RT-qPCR were significantly upregulated in full maturation and showed the highest variability at véraison and the lowest gene expression values in the pea size stageinfo:eu-repo/semantics/publishedVersio

Polyclonal selection for abiotic stress tolerance in Arinto: Implications in yield and quality of the must

The valorisation of genetic variability through the identification suitable genotypes for traits such as yield and must quality is an effective strategy used for grapevine selection. Currently, climate change-driven heat waves and drought affect plant growth and wine quality, but little information is available on intravarietal variability regarding responses to stress. In the current work, the intravarietal genetic variability of the Portuguese variety Arinto was studied for yield, must quality, and tolerance to abiotic stress. An innovative approach using rapid, and nondestructive measurements of surface leaf temperature (SLT), Normalized Difference Vegetation Index (NDVI), Photochemical Reflectance Index (PRI), and chlorophyll content (SPAD), was used in an experimental population of 165 clones of Arinto installed according to a resolvable row-column design with 6 replicates. Also, yield and quality characteristics of the must were quantified. Linear mixed models were fitted to the data, and the empirical best linear unbiased predictors (EBLUPs) of genotypic effects for each trait were obtained as well as the coefficient of genotypic variation (CVG) and broad sense heritability. The results enabled the selection of a group of genotypes with increased tolerance to stress, which maintained the must quality of Arinto

Proof-of-Concept Study of Multifunctional Hybrid Nanoparticle System Combined with NIR Laser Irradiation for the Treatment of Melanoma

Supplementary Materials - available online at https://www.mdpi.com/article/10.3390/biom11040511/s1The global impact of cancer emphasizes the importance of developing innovative, effective and minimally invasive therapies. In the context of superficial cancers, the development of a multifunctional nanoparticle-based system and its in vitro and in vivo safety and efficacy characterization are, herein, proposed as a proof-of-concept. This multifunctional system consists of gold nanoparticles coated with hyaluronic and oleic acids, and functionalized with epidermal growth factor for greater specificity towards cutaneous melanoma cells. This nanoparticle system is activated by a near-infrared laser. The characterization of this nanoparticle system included several phases, with in vitro assays being firstly performed to assess the safety of gold nanoparticles without laser irradiation. Then, hairless immunocompromised mice were selected for a xenograft model upon inoculation of A375 human melanoma cells. Treatment with near-infrared laser irradiation for five minutes combined with in situ administration of the nanoparticles showed a tumor volume reduction of approximately 80% and, in some cases, led to the formation of several necrotic foci, observed histologically. No significant skin erythema at the irradiation zone was verified, nor other harmful effects on the excised organs. In conclusion, these assays suggest that this system is safe and shows promising results for the treatment of superficial melanoma.The authors would like to thank to Fundação para a Ciência e Tecnologia (FCT) for the essential financial support under the project’s references PTDC/BBB-BMC/0611/2012, UIDB/00645/2020, UIDB/04138/2020 and UIDP/04138/2020 as well as for the PhD fellowships SFRH/BD/148044/2019 and SFRH/BD/147306/2019.info:eu-repo/semantics/publishedVersio

Proof-of-Concept Study of Multifunctional Hybrid Nanoparticle System Combined with NIR Laser Irradiation for the Treatment of Melanoma

Abstract: The global impact of cancer emphasizes the importance of developing innovative, effective and minimally invasive therapies. In the context of superficial cancers, the development of a multifunctional nanoparticle-based system and its in vitro and in vivo safety and efficacy characterization are, herein, proposed as a proof-of-concept. This multifunctional system consists of gold nanoparticles coated with hyaluronic and oleic acids, and functionalized with epidermal growth factor for greater specificity towards cutaneous melanoma cells. This nanoparticle system is activated by a near-infrared laser. The characterization of this nanoparticle system included several phases, with in vitro assays being firstly performed to assess the safety of gold nanoparticles without laser irradiation. Then, hairless immunocompromised mice were selected for a xenograft model upon inoculation of A375 human melanoma cells. Treatment with near-infrared laser irradiation for five minutes combined with in situ administration of the nanoparticles showed a tumor volume reduction of approximately 80% and, in some cases, led to the formation of several necrotic foci, observed histologically. No significant skin erythema at the irradiation zone was verified, nor other harmful effects on the excised organs. In conclusion, these assays suggest that this system is safe and shows promising results for the treatment of superficial melanoma

Large-scale screening of unknown varieties in a grapevine intra-varietal variability collection

Since the last decade of the last century, it is known that many old grapevine varieties are descendants of other varieties through natural crossing. Portugal has an important program for the conservation of representative samples of intra-varietal variability of all autochthonous varieties, managed by the Portuguese Association for Grapevine Diversity (PORVID), which makes looking for genotypes with dubious identification an important activity from a perspective of its valorisation. This communication presents the results of the molecular analysis of 5,000 samples (accessions) from the PORVID’s collection, using nine microsatellite loci currently recommended by the International Organization of Vine and Wine (OIV) for genetic grapevine identification. The results obtained confirmed the molecular identity of 4,220 samples corresponding to 214 varieties present in the official list of Portuguese varieties. In 780 samples, 95 profiles with a plural number of accessions revealed not to be listed in the Vitis International Variety Catalogue (VIVC) database, corresponding to possible varieties either descendent from natural crossing from at least one known parental variety, or from undetermined origin. Furthermore, the need for a comprehensive strategy aimed at uncovering other hidden varieties is discussed to prevent their imminent loss, deepen understanding of their origin, and add economic value and sustainability to the vine and wine sector

Polyclonal selection for abiotic stress tolerance in Arinto: Implications in yield and quality of the must

The valorisation of genetic variability through the identification suitable genotypes for traits such as yield and must quality is an effective strategy used for grapevine selection. Currently, climate change-driven heat waves and drought affect plant growth and wine quality, but little information is available on intravarietal variability regarding responses to stress. In the current work, the intravarietal genetic variability of the Portuguese variety Arinto was studied for yield, must quality, and tolerance to abiotic stress. An innovative approach using rapid, and nondestructive measurements of surface leaf temperature (SLT), Normalized Difference Vegetation Index (NDVI), Photochemical Reflectance Index (PRI), and chlorophyll content (SPAD), was used in an experimental population of 165 clones of Arinto installed according to a resolvable row-column design with 6 replicates. Also, yield and quality characteristics of the must were quantified. Linear mixed models were fitted to the data, and the empirical best linear unbiased predictors (EBLUPs) of genotypic effects for each trait were obtained as well as the coefficient of genotypic variation (CVG) and broad sense heritability. The results enabled the selection of a group of genotypes with increased tolerance to stress, which maintained the must quality of Arinto

Modulation of the Berry Skin Transcriptome of cv. Tempranillo Induced by Water Stress Levels

Climate change in the Mediterranean area is making summers warmer and dryer. Grapevine (Vitis vinifera L.) is mostly important for wine production in Mediterranean countries, and the variety Tempranillo is one of the most cultivated in Spain and Portugal. Drought decreases yield and quality and causes important economic losses. As full irrigation has negative effects on quality and water is scarce in this region, deficit irrigation is often applied. In this research, we studied the effects of two deficit irrigation treatments, Sustained Deficit Irrigation (SDI) and Regulated Deficit Irrigation (RDI), on the transcriptome of grape berries at full maturation, through RNAseq. The expression of differentially regulated genes (DEGs) was also monitored through RT-qPCR along berry development. Most transcripts were regulated by water stress, with a similar distribution of up- and down-regulated transcripts within functional categories (FC). Primary metabolism was the more severely affected FC under water stress, followed by signaling and transport. Almost all DEGs monitored were significantly up-regulated by severe water stress at veraison. The modulation of an auxin response repression factor, AUX22D, by water stress indicates a role of this gene in the response to drought. Further, the expression of WRKY40, a TF that regulates anthocyanin biosynthesis, may be responsible for changes in grape quality under severe water stress

Large-scale screening of unknown varieties in a grapevine intra-varietal variability collection

Since the last decade of the last century, it is known that many old grapevine varieties are descendants of other varieties through natural crossing. Portugal has an important program for the conservation of representative samples of intra-varietal variability of all autochthonous varieties, managed by the Portuguese Association for Grapevine Diversity (PORVID), which makes looking for genotypes with dubious identification an important activity from a perspective of its valorisation. This communication presents the results of the molecular analysis of 5,000 samples (accessions) from the PORVID’s collection, using nine microsatellite loci currently recommended by the International Organization of Vine and Wine (OIV) for genetic grapevine identification. The results obtained confirmed the molecular identity of 4,220 samples corresponding to 214 varieties present in the official list of Portuguese varieties. In 780 samples, 95 profiles with a plural number of accessions revealed not to be listed in the Vitis International Variety Catalogue (VIVC) database, corresponding to possible varieties either descendent from natural crossing from at least one known parental variety, or from undetermined origin. Furthermore, the need for a comprehensive strategy aimed at uncovering other hidden varieties is discussed to prevent their imminent loss, deepen understanding of their origin, and add economic value and sustainability to the vine and wine sector