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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.

Worldwide, extensive agricultural losses are attributed to drought, often in combination with heat in Mediterranean climate regions, where grapevine traditionally grows. The available scenarios for climate change suggest increases in aridity in these regions. Under natural conditions plants are affected by a combination of stresses, triggering synergistic or antagonistic physiological, metabolic or transcriptomic responses unique to the combination. However the study of such stresses in a controlled environment can elucidate important mechanisms by allowing the separation of the effects of individual stresses. To gather those effects, cuttings of two grapevine varieties, Touriga Nacional ( TN) and Trincadeira ( TR), were grown under controlled conditions and subjected to three abiotic stresses (drought - WS, heat - HS and high light - LS) individually and in combination two-by-two ( WSHS, WSLS, HSLS) or all three ( WSHSLS). Photosynthesis, water status, contents of H2O2, abscisic acid and metabolites of the ascorbate-glutathione cycle were measured in the leaves. Common and distinct response features were identified in the different stress combinations. Photosynthesis was not hindered in TN by LS, while even individual stresses severely affect photosynthesis in TR. Abscisic acid may be implicated in grapevine osmotic responses since it is correlated with tolerance parameters, especially in combined stresses involving drought. Overall, the responses to drought-including treatments were clearly distinct to those without drought. From the specific behaviours of the varieties, it can be concluded that TN shows a higher capacity for heat dissipation and for withstanding high light intensities, indicating better adjustment to warm conditions, provided that water supply is plentiful. [ABSTRACT FROM AUTHOR]

Extensive agricultural losses are attributed to heat, often combined with drought. These abiotic stresses occur in the field simultaneously, namely in areas with Mediterranean climate, where grapevine traditionally grows. The available scenarios for climate change suggest an increase in the frequency of heat waves and severe drought events in summer, also affecting the South of Portugal. In this work we monitored several production-related parameters and evaluated the state of the oxidative stress response apparatus of two grapevine varieties, Touriga Nacional (TN) and Trincadeira (TR), with and without irrigation, during three field seasons (2010 to 2012). Overall, results point to a high correlation of most yield and stress-associated parameters with the specific characteristics of each variety and to each season rather than the irrigation treatments. In the season with the driest winter, 2012, the lack of irrigation significantly affected yield in TR, while in the two other seasons the impact of the irrigation regime was much lower. In 2012, the yield of TN was affected by environmental conditions of the previous season. The irrigation treatments significantly affected berry size rather than quality.

Plant protein expression can be a challenging enterprise in any biochemical or molecular biology research project. Several heterologous systems like bacteria, yeast, insect cells and cell free systems have been used to produce plant proteins for in vitro experiments and structural characterization. However, due to particularities of plant proteins, for example the specific type and abundance of post-translational modifications (e.g. glycosylation), a plant system to express plant proteins is extremely desirable. The use of Nicotiana benthamiana (N. benthamiana) plants for protein expression has proven to be quick and reliable. To illustrate the robustness and rapidity of this system, recent efforts to produce the first protein based drug against the Ebola virus was conducted in N. benthamiana protein expression systems (Choi et al., 2015). This protocol describes a simple system for the expression and enrichment (affinity purification) of plant apoplastic proteins in N. benthamiana leaves, which was successfully used in the characterization of the Arabidopsis thaliana pectin acetylesterases, PAE8 and PAE9 (de Souza et al., 2014).

Abstract Background Quantitative real time polymerase chain reaction is becoming the primary tool for detecting mRNA and transcription data analysis as it shows to have advantages over other more commonly used techniques. Nevertheless, it also presents a few shortcomings, with the most import being the need for data normalisation, usually with a reference gene. Therefore the choice of the reference gene(s) is of great importance for correct data analysis. Microarray data, when available, can be of great assistance when choosing reference genes. Grapevine was submitted to water stress and heat stress as well as a combination of both to test the stability of the possible reference genes. Results Using the analysis of microarray data available for grapevine, six possible reference genes were selected for RT-qPCR validation: PADCP, ubiq, TIF, TIF-GTP, VH1-IK, aladin-related. Two additional genes that are commonly used as reference genes were included: act and L2. The stability of those genes was tested in leaves of grapevine in both field plants and in greenhouse plants under water or heat stress or a combination of both. Gene stability was analyzed with the softwares GeNorm, NormFinder and the ΔCq method resulting in several combinations of reference genes suitable for data normalisation. In order to assess the best combination, the reference genes were tested in putative stress marker genes (PCO, Galsynt, BKCoAS and HSP17) also chosen from the same microarray, in water stress, heat stress and the combination of both. Conclusions Each method selected different gene combinations (PADCP + act, TIF + TIF-GTP and ubiq + act). However, as none of the combinations diverged significantly from the others used to normalize the expression of the putative stress marker genes, then any combination is suitable for data normalisation under the conditions tested. Here we prove the accuracy of choosing grapevine reference genes for RT-qPCR through a microarray analysis.

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.

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 stage.

High temperatures and extreme drought are increasingly more frequent in Portugal, which represents a strong threat to viticulture in certain regions of the country. These multifactorial abiotic stresses are threatening viticultural areas worldwide, and the problem can hardly be overcome only by changing cultural practices. This scenario has raised a major challenge for plant scientists to find ways to adapt existing varieties to the new conditions without loss of their characteristic flavors, yield, and associated varietal character of wines. Aragonez (syn. Tempranillo) is one such variety, widely cultivated in Portugal and Spain, with specific characteristics associated with terroir . In this context, insight into intravarietal variability to enable its exploitation for selection becomes an important tool to mitigate the effect of multifactorial stresses driven by climate changes. The present work describes an innovative selection approach: selection for abiotic stress tolerance, measured by the leaf temperature of clones under environmental conditions of drought and extreme heat. This evaluation was complemented with values of yield and quality characteristics of the must (pH, acidity, °Brix, and anthocyanins). The application of this methodology was done in an experimental population of 255 clones of Tempranillo for 3 years. The genotypes were then ranked according to their level of tolerance to abiotic stress without loss of yield/quality. To understand the differences at the transcription level that could account for such variability, several of the most tolerant and most sensitive genotypes were analyzed for key genes using reverse transcriptase-quantitative polymerase chain reaction. The results enabled the selection of a group of genotypes with increased tolerance to stress, in relation to the average of the variety, which maintained the typical must quality of Aragonez. In parallel, several transcripts previously acknowledged as markers for abiotic stress tolerance were identified in several clones and are possible targets for plant breeding and genetic modification and/or to develop screening procedures to select genotypes better adapted to the abiotic stress driven by climate change., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Carvalho, Gonçalves, Amâncio and Martins.)

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.

Vitis vinifera can be divided into two subspecies, V. vinifera subsp. vinifera , one of the most important agricultural crops in the world, and its wild ancestor, V. vinifera subsp. sylvestris . Three flower types can be observed: hermaphrodite and female (on some varieties) in vinifera , and male or female flowers in sylvestris . It is assumed that the different flower types in the wild ancestor arose through specific floral patterns of organ abortion. A considerable amount of data about the diversity of sexual systems in grapevines has been collected over the past century. Several grapevine breeding studies led to the hypothesis that dioecy in vinifera is derived from a hermaphrodite ancestor and could be controlled by either, one or two linked genetic determinants following Mendelian inherence. More recently, experiments using molecular approaches suggested that these loci were located in a specific region of the chromosome 2 of vinifera . Based on the works published so far, its seems evident that a putative sex locus is present in chromosome 2. However, it is still not fully elucidated whether flower types are regulated by two linked loci or by one locus with three alleles. Nevertheless, several genes could contribute to sex determination in grapevine. This review presents the results from early studies, combined with the recent molecular approaches, which may contribute to the design of new experiments towards a better understanding of the sex inheritance in grapevine., Competing Interests: The authors declare that they have no competing interests., (© 2019 Coito et al.)

In Mediterranean climate areas, the available scenarios for climate change suggest an increase in the frequency of heat waves and severe drought in summer. Grapevine (Vitis vinifera L.) is a traditional Mediterranean species and is the most valuable fruit crop in the world. Currently, viticulture must adjust to impending climate changes that are already pushing vine-growers toward the use of irrigation, with the concomitant losses in wine quality, and researchers to study tolerance to stress in existing genotypes. The viticulture and winemaking worlds are in demand to understand the physiological potential of the available genotypes to respond to climate changes. In this review, we will focus on the cross-talk between common abiotic stresses that currently affect grapevine productivity and that are prone to affect it deeper in the future. We will discuss results obtained under three experimental stress conditions and that call for specific responses: (1) acclimatization of in vitro plantlets, (2) stress combinations in controlled conditions for research purposes, (3) extreme events in the field that, driven by climate changes, are pushing Mediterranean species to the limit. The different levels of tolerance to stress put in evidence by the plasticity of phenotypic and genotypic response mechanisms, will be addressed. This information is relevant to understand varietal adaptation to impending climate changes and to assist vine growers in choosing genotypes and viticulture practices., (© 2018 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

The most discriminating characteristic between the cultivated Vitis vinifera subsp. vinifera and the wild-form Vitis vinifera subsp. sylvestris is their sexual system. Flowers of cultivars are mainly hermaphroditic, whereas wild plants have female and male individuals whose flowers follow a hermaphroditic pattern during early stages of development and later develop non-functional reproductive organs. In angiosperms, the basic developmental system for floral organ identity is explained by the ABCDE model. This model postulates that regulatory gene functions work in a combinatorial way to confer organ identity in each whorl. In wild Vitis nothing is known about the function and expression profile of these genes. Here we show an overall view of the temporal and spatial expression pattern of the ABCDE genes as well as the pattern of VviSUPERMAN that establishes a boundary between the stamen and the carpel whorls, in the male, female and complete flower types. The results show a similar pattern in Vitis species suggesting that the pathway leading to unisexuality acts independently and/or downstream of B- and C- function genes.

Widespread agricultural losses attributed to drought, often combined with high temperatures, frequently occur in the field, namely in Mediterranean climate areas, where the existing scenarios for climate change indicate an increase in the frequency of heat waves and severe drought events in summer. Grapevine ( Vitis vinifera L.) is the most cultivated fruit species in the world and the most valuable one and is a traditional Mediterranean species. Currently, viticulture must adjust to impending climate changes that are already pushing vine-growers toward the use of ancient and resilient varieties. Portugal is very rich in grapevine biodiversity, however, currently, 90% of the total producing area is planted with only 16 varieties. There is a pressing need to understand the existing genetic diversity and the physiological potential of the varieties/genotypes available to be able to respond to climate changes. With the above scenario in mind, an assembly of 65 differentially expresses genes (DEGs) previously identified as responsive to abiotic stresses in two well studied genotypes, 'Touriga Nacional' and 'Trincadeira,' was designed to scan the gene expression of leaf samples from 10 traditional Portuguese varieties growing in two regions with distinct environmental conditions. Forty-five of those DEGs proved to be associated to "abiotic stress" and were chosen to build a custom qPCR array to identify uncharacterized genotypes as sensitive or tolerant to abiotic stress. According to the experimental set-up behind the array design these DEGs can also be used as indicators of the main abiotic stress that the plant is subjected and responding to (drought, heat, or excess light).

The noteworthy fine remodeling that plant cell walls (CWs) undergo to adapt to developmental, physiological and environmental cues and the observation that its composition and dynamics differ between species represents an opportunity to couple crop species agronomic studies with research on CW modifications. Vitis vinifera is one of the most important crops from an economic point-of-view due to the high value of the fruit, predominantly for winemaking. The availability of some information related to this species' CWs allows researching its responses to imposed conditions that affect the plant's development. Mineral deficiency, in particular nitrogen, phosphorus, potassium and sulfur, strongly affects plant metabolism, reducing both growth and crop yield. Despite the importance of mineral nutrition in development, its influence on CW synthesis and modifications is still insufficiently documented. Addressing this knowledge gap, V. vinifera experimental models were used to study CW responses to imposed mineral depletion in unorganized (callus) and organized (shoots) tissues. The discussion of the obtained results is the main focus of this review. Callus and shoots submitted to mineral restriction are impaired in specific CW components, predominantly cellulose. Reorganization on structure and deposition of several other polymers, in particular the degree and pattern of pectin methyl-esterification and the amount of xyloglucan (XyG), arabinan and extensin, is also observed. In view of recently proposed CW models that consider biomechanical hotspots and direct linkages between pectins and XyG/cellulose, the outcome of these modifications in explaining maintenance of CW integrity through compensatory stiffening can be debated. Nutrient stresses do not affect evenly all tissues with undifferentiated callus tissues showing more pronounced responses, followed by shoot mature internodes, and then newly formed internodes. The impact of nitrogen depletion leads to more noticeable responses, supporting this nutrient's primary role in plant development and metabolism. The consequential compensatory mechanisms highlight the pivotal role of CW in rearranging under environmental stresses.

Vitis vinifera vinifera is a hermaphrodite subspecies, while its ancestor, Vitis vinifera sylvestris , is dioecious. We have identified two genes that together allow the discrimination between male, female and hermaphrodite Vitis plants. The sex locus region on chromosome 2 was screened resulting in the discovery of a new gene, VviFSEX . The same screening revealed another gene, VviAPRT3 , located in the sex region, that be used as a sex marker. Both genes are good candidates to be involved in flower sex differentiation in grapevine. To assess their role in sex specification, spatial and temporal expression analysis was performed. The expression of VviFSEX is detected in petals, stamens and carpel primordia of all flower types, making its putative function unclear; however, female plants display a single allele for this gene, while male and hermaphrodites display two alleles. On the other hand, the specific expression of VviAPRT3 in the carpel primordial of male plants suggests a possible role in the abortion of pistil structures. We propose a model to explain the carpel abortion in male flowers and the absence of stamen viability in female flowers. In addition, this work reinforces the presence of a sex locus on Vitis chromosome 2.

Key Message: RNA-seq of Vitis during early stages of bud development, in male, female and hermaphrodite flowers, identified new loci outside of annotated gene models, suggesting their involvement in sex establishment. The molecular mechanisms responsible for flower sex specification remain unclear for most plant species. In the case of V. vinifera ssp. vinifera, it is not fully understood what determines hermaphroditism in the domesticated subspecies and male or female flowers in wild dioecious relatives (Vitis vinifera ssp. sylvestris). Here, we describe a de novo assembly of the transcriptome of three flower developmental stages from the three Vitis vinifera flower types. The validation of de novo assembly showed a correlation of 0.825. The main goals of this work were the identification of V. v. sylvestris exclusive transcripts and the characterization of differential gene expression during flower development. RNA from several flower developmental stages was used previously to generate Illumina sequence reads. Through a sequential de novo assembly strategy one comprehensive transcriptome comprising 95,516 non-redundant transcripts was assembled. From this dataset 81,064 transcripts were annotated to V. v. vinifera reference transcriptome and 11,084 were annotated against V. v. vinifera reference genome. Moreover, we found 3368 transcripts that could not be mapped to Vitis reference genome. From all the non-redundant transcripts that were assembled, bioinformatics analysis identified 133 specific of V. v. sylvestris and 516 transcripts differentially expressed among the three flower types. The detection of transcription from areas of the genome not currently annotated suggests active transcription of previously unannotated genomic loci during early stages of bud development.

Background: Predicted climate changes announce an increase of extreme environmental conditions including drought and excessive heat and light in classical viticultural regions. Thus, understanding how grapevine responds to these conditions and how different genotypes can adapt, is crucial for informed decisions on accurate viticultural actions. Global transcriptome analyses are useful for this purpose as the response to these abiotic stresses involves the interplay of complex and diverse cascades of physiological, cellular and molecular events. The main goal of the present work was to evaluate the response to diverse imposed abiotic stresses at the transcriptome level and to compare the response of two grapevine varieties with contrasting physiological trends, Trincadeira (TR) and Touriga Nacional (TN)., Results: Leaf transcriptomic response upon heat, high light and drought treatments in growth room controlled conditions, as well as full irrigation and non-irrigation treatments in the field, was compared in TR and TN using GrapeGene GeneChips®. Breakdown of metabolism in response to all treatments was evidenced by the functional annotation of down-regulated genes. However, circa 30 % of the detected stress-responsive genes are still annotated as «Unknown» function. Selected differentially expressed genes from the GrapeGene GeneChip® were analysed by RT-qPCR in leaves of growth room plants under the combination of individual stresses and of field plants, in both varieties. The transcriptomic results correlated better with those obtained after each individual stress than with the results of plants from field conditions., Conclusions: From the transcriptomic comparison between the two Portuguese grapevine varieties Trincadeira and Touriga Nacional under abiotic stress main conclusions can be drawn: 1. A different level of tolerance to stress is evidenced by a lower transcriptome reprogramming in TN than in TR. Interestingly, this lack of response in TN associates with its higher adaptation to extreme conditions including environmental conditions in a changing climate; 2. A complex interplay between stress transcriptional cascades is evidenced by antagonistic and, in lower frequency, synergistic effects on gene expression when several stresses are imposed together; 3. The grapevine responses to stress under controlled conditions are not fully extrapolated to the complex vineyard scenario and should be cautiously considered for agronomic management decision purposes.

Cell wall (CW) is a dynamic structure that determines the plant form, growth and response to environmental conditions. Vitis vinifera callus grown under nitrogen (-N), phosphorous (-P) and sulfur (-S) deficiency were used as a model system to address the influence of mineral stress in CW remodeling. Callus cells morphology was altered, mostly under -N, resulting in changes in cell length and width compared with the control. CW composition ascertained with specific staining and immuno-detection showed a decrease in cellulose and altered pattern of pectin methylesterification. Under mineral stress genes expression from candidate families disclosed mainly a downregulation of a glycosyl hydrolase family 9C (GH9C), xyloglucan transglycosylase/hydrolases (XTHs) with predicted hydrolytic activity and pectin methylesterases (PMEs). Conversely, upregulation of PMEs inhibitors (PMEIs) was observed. While methylesterification patterns can be associated to PME/PMEI gene expression, the lower cellulose content cannot be attributed to altered cellulose synthase (CesA) gene expression suggesting the involvement of other gene families. Salt extracts from -N and -P callus tissues increased plastic deformation in cucumber hypocotyls while no effect was observed with -S extracts. The lower endo-acting glycosyl hydrolase activity of -N callus extracts pinpoints a more expressive impact of -N on CW-remodeling., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Peroxiredoxins (Prx) catalyse the reduction of hydrogen peroxide (H2O2) and, in association with catalases and other peroxidases, may participate in signal transduction by regulating intercellular H2O2 concentration that in turn can control gene transcription and cell signaling. Using virus-induced-gene-silencing (VIGS), 2-Cys Peroxiredoxin (2CysPrx) family and type-II Peroxiredoxin B (PrxIIB) gene were silenced in Nicotiana benthamiana, to study the impact that the loss of function of each Prx would have in the antioxidant system under control (22 °C) and severe heat stress conditions (48 °C). The results showed that both Prxs, although in different organelles, influence the regeneration of ascorbate to a significant extent, but with different purposes. 2CysPrx affects abscisic acid (ABA) biosynthesis through ascorbate, while PrxIIB does it probably through the xanthophyll cycle. Moreover, 2CysPrx is key in H2O2 scavenging and in consequence in the regulation of ABA signaling downstream of reactive oxygen species and PrxIIB provides an important assistance for H2O2 peroxisome scavenges., (© 2014 Institute of Botany, Chinese Academy of Sciences.)