Transcriptomic analysis of temporal shifts in berry development between two grapevine cultivars of the Pinot family reveals potential genes controlling ripening time.

BACKGROUND: Grapevine cultivars of the Pinot family represent clonally propagated mutants with major phenotypic and physiological differences, such as different colour or shifted ripening time, as well as changes in important viticultural traits. Specifically, the cultivars 'Pinot Noir' (PN) and 'Pinot Noir Precoce' (PNP, early ripening) flower at the same time, but vary in the beginning of berry ripening (veraison) and, consequently, harvest time. In addition to genotype, seasonal climatic conditions (i.e. high temperatures) also affect ripening times. To reveal possible regulatory genes that affect the timing of veraison onset, we investigated differences in gene expression profiles between PN and PNP throughout berry development with a closely meshed time series and over two separate years. RESULTS: The difference in the duration of berry formation between PN and PNP was quantified to be approximately two weeks under the growth conditions applied, using plant material with a proven PN and PNP clonal relationship. Clusters of co-expressed genes and differentially expressed genes (DEGs) were detected which reflect the shift in the timing of veraison onset. Functional annotation of these DEGs fit to observed phenotypic and physiological changes during berry development. In total, we observed 3,342 DEGs in 2014 and 2,745 DEGs in 2017 between PN and PNP, with 1,923 DEGs across both years. Among these, 388 DEGs were identified as veraison-specific and 12 were considered as berry ripening time regulatory candidates. The expression profiles revealed two candidate genes for ripening time control which we designated VviRTIC1 and VviRTIC2 (VIT_210s0071g01145 and VIT_200s0366g00020, respectively). These genes likely contribute the phenotypic differences observed between PN and PNP. CONCLUSIONS: Many of the 1,923 DEGs show highly similar expression profiles in both cultivars if the patterns are aligned according to developmental stage. In our work, putative genes differentially expressed between PNP and PN which could control ripening time as well as veraison-specific genes were identified. We point out connections of these genes to molecular events during berry development and discuss potential candidate genes which may control ripening time. Two of these candidates were observed to be differentially expressed in the early berry development phase. Several down-regulated genes during berry ripening are annotated as auxin response factors / ARFs. Conceivably, general changes in auxin signaling may cause the earlier ripening phenotype of PNP.

High-Precision Phenotyping of Grape Bunch Architecture Using Fast 3D Sensor and Automation.

Wine growers prefer cultivars with looser bunch architecture because of the decreased risk for bunch rot. As a consequence, grapevine breeders have to select seedlings and new cultivars with regard to appropriate bunch traits. Bunch architecture is a mosaic of different single traits which makes phenotyping labor-intensive and time-consuming. In the present study, a fast and high-precision phenotyping pipeline was developed. The optical sensor Artec Spider 3D scanner (Artec 3D, L-1466, Luxembourg) was used to generate dense 3D point clouds of grapevine bunches under lab conditions and an automated analysis software called 3D-Bunch-Tool was developed to extract different single 3D bunch traits, i.e., the number of berries, berry diameter, single berry volume, total volume of berries, convex hull volume of grapes, bunch width and bunch length. The method was validated on whole bunches of different grapevine cultivars and phenotypic variable breeding material. Reliable phenotypic data were obtained which show high significant correlations (up to r² = 0.95 for berry number) compared to ground truth data. Moreover, it was shown that the Artec Spider can be used directly in the field where achieved data show comparable precision with regard to the lab application. This non-invasive and non-contact field application facilitates the first high-precision phenotyping pipeline based on 3D bunch traits in large plant sets.

Phenoliner: A New Field Phenotyping Platform for Grapevine Research.

In grapevine research the acquisition of phenotypic data is largely restricted to the field due to its perennial nature and size. The methodologies used to assess morphological traits and phenology are mainly limited to visual scoring. Some measurements for biotic and abiotic stress, as well as for quality assessments, are done by invasive measures. The new evolving sensor technologies provide the opportunity to perform non-destructive evaluations of phenotypic traits using different field phenotyping platforms. One of the biggest technical challenges for field phenotyping of grapevines are the varying light conditions and the background. In the present study the Phenoliner is presented, which represents a novel type of a robust field phenotyping platform. The vehicle is based on a grape harvester following the concept of a moveable tunnel. The tunnel it is equipped with different sensor systems (RGB and NIR camera system, hyperspectral camera, RTK-GPS, orientation sensor) and an artificial broadband light source. It is independent from external light conditions and in combination with artificial background, the Phenoliner enables standardised acquisition of high-quality, geo-referenced sensor data.

LRP2, an auxiliary receptor that controls sonic hedgehog signaling in development and disease.

To fulfill their multiple roles in organ development and adult tissue homeostasis, hedgehog (HH) morphogens act through their receptor Patched (PTCH) on target cells. However, HH actions also require HH binding proteins, auxiliary cell surface receptors that agonize or antagonize morphogen signaling in a context-dependent manner. Here, we discuss recent findings on the LDL receptor-related protein 2 (LRP2), an exemplary HH binding protein that modulates sonic hedgehog activities in stem and progenitor cell niches in embryonic and adult tissues. LRP2 functions are crucial for developmental processes in a number of tissues, including the brain, the eye, and the heart, and defects in this receptor pathway are the cause of devastating congenital diseases in humans. Developmental Dynamics 245:569-579, 2016. © 2016 Wiley Periodicals, Inc.

Hyperspectral phenotyping of the reaction of grapevine genotypes to Plasmopara viticola.

A major aim in grapevine breeding is the provision of cultivars resistant to downy mildew. As Plasmopara viticola produces sporangia on the abaxial surface of susceptible cultivars, disease symptoms on both leaf sides may be detected and quantified by technical sensors. The response of cultivars 'Mueller-Thurgau', 'Regent', and 'Solaris', which differ in resistance to P. viticola, was characterized under controlled conditions by using hyperspectral sensors. Spectral reflectance was suitable to differentiate between non-infected cultivars and leaf sides of the bicolored grapevine. Brown discoloration of tissue became visible on both leaf sides of resistant cultivars 2 days before downy mildew symptoms appeared on the susceptible 'Mueller-Thurgau' cultivar. Infection of this cultivar resulted in significant (P<0.05) reflectance changes 1-2 days prior to abaxial sporulation induced by high relative humidity, or the formation of adaxial oil spots. Hyperspectral imaging was more sensitive in disease detection than non-imaging and provided spatial information on the leaf level. Spectral indices provided information on the variability of chlorophyll content, photosynthetic activity, and relative water content of leaf tissue in time and space. On 'Mueller-Thurgau' downy mildew translated reflectance to higher values as detectable by the index DMI_3=(R470+R682+R800)/(R800/R682) and affected reflectance at 1450nm. Tissue discoloration on 'Regent' and 'Solaris' cultivars was associated with lower reflectance between 750 and 900nm; blue and red reflectance demonstrated differences from leaf necroses. With high inoculum densities, P. viticola sporulated on even resistant cultivars. Hyperspectral characterization at the tissue level proved suitable for phenotyping plant resistance to pathogens and provided information on resistance mechanisms.

Field phenotyping of grapevine growth using dense stereo reconstruction.

BACKGROUND: The demand for high-throughput and objective phenotyping in plant research has been increasing during the last years due to large experimental sites. Sensor-based, non-invasive and automated processes are needed to overcome the phenotypic bottleneck, which limits data volumes on account of manual evaluations. A major challenge for sensor-based phenotyping in vineyards is the distinction between the grapevine in the foreground and the field in the background - this is especially the case for red-green-blue (RGB) images, where similar color distributions occur both in the foreground plant and in the field and background plants. However, RGB cameras are a suitable tool in the field because they provide high-resolution data at fast acquisition rates with robustness to outdoor illumination. RESULTS: This study presents a method to segment the phenotypic classes 'leaf', 'stem', 'grape' and 'background' in RGB images that were taken with a standard consumer camera in vineyards. Background subtraction is achieved by taking two images of each plant for depth reconstruction. The color information is furthermore used to distinguish the leaves from stem and grapes in the foreground. The presented approach allows for objective computation of phenotypic traits like 3D leaf surface areas and fruit-to-leaf ratios. The method has been successfully applied to objective assessment of growth habits of new breeding lines. To this end, leaf areas of two breeding lines were monitored and compared with traditional cultivars. A statistical analysis of the method shows a significant (p <0.001) determination coefficient R (2)= 0.93 and root-mean-square error of 3.0%. CONCLUSIONS: The presented approach allows for non-invasive, fast and objective assessment of plant growth. The main contributions of this study are 1) the robust segmentation of RGB images taken from a standard consumer camera directly in the field, 2) in particular, the robust background subtraction via reconstruction of dense depth maps, and 3) phenotypic applications to monitoring of plant growth and computation of fruit-to-leaf ratios in 3D. This advance provides a promising tool for high-throughput, automated image acquisition, e.g., for field robots.

Impedance of the grape berry cuticle as a novel phenotypic trait to estimate resistance to Botrytis cinerea.

Warm and moist weather conditions during berry ripening provoke Botrytis cinerea (B. cinerea) causing notable bunch rot on susceptible grapevines with the effect of reduced yield and wine quality. Resistance donors of genetic loci to increase B. cinerea resistance are widely unknown. Promising traits of resistance are represented by physical features like the thickness and permeability of the grape berry cuticle. Sensor-based phenotyping methods or genetic markers are rare for such traits. In the present study, the simple-to-handle I-sensor was developed. The sensor enables the fast and reliable measurement of electrical impedance of the grape berry cuticles and its epicuticular waxes (CW). Statistical experiments revealed highly significant correlations between relative impedance of CW and the resistance of grapevines to B. cinerea. Thus, the relative impedance Zrel of CW was identified as the most important phenotypic factor with regard to the prediction of grapevine resistance to B. cinerea. An ordinal logistic regression analysis revealed a R2McFadden of 0.37 and confirmed the application of Zrel of CW for the prediction of bunch infection and in this way as novel phenotyping trait. Applying the I-sensor, a preliminary QTL region was identified indicating that the novel phenotypic trait is as well a valuable tool for genetic analyses.

An automated field phenotyping pipeline for application in grapevine research.

Due to its perennial nature and size, the acquisition of phenotypic data in grapevine research is almost exclusively restricted to the field and done by visual estimation. This kind of evaluation procedure is limited by time, cost and the subjectivity of records. As a consequence, objectivity, automation and more precision of phenotypic data evaluation are needed to increase the number of samples, manage grapevine repositories, enable genetic research of new phenotypic traits and, therefore, increase the efficiency in plant research. In the present study, an automated field phenotyping pipeline was setup and applied in a plot of genetic resources. The application of the PHENObot allows image acquisition from at least 250 individual grapevines per hour directly in the field without user interaction. Data management is handled by a database (IMAGEdata). The automatic image analysis tool BIVcolor (Berries in Vineyards-color) permitted the collection of precise phenotypic data of two important fruit traits, berry size and color, within a large set of plants. The application of the PHENObot represents an automated tool for high-throughput sampling of image data in the field. The automated analysis of these images facilitates the generation of objective and precise phenotypic data on a larger scale.

Deep incorporation of organic amendments into soils of a 'Calardis Musqué' vineyard: effects on greenhouse gas emissions, vine vigor, and grape quality.

Background: Traditional wine growing regions are increasingly endangered by climatic alterations. One promising approach to mitigate advancing climate change could be an increase of soil organic matter. Here, especially subsoils are of interest as they provide higher carbon storage potential than topsoils. In this context, vineyard subsoils could be particularly suitable since they are deeply cultivated once before planting and afterwards, left at rest for several decades due to the perennial nature of grapevines. Methods: For this purpose, a biochar compost substrate and greenwaste compost were incorporated in up to 0.6 m depth before planting a new experimental vineyard with the fungus-resistant grapevine cultivar 'Calardis Musqué'. The influence of this deep incorporation on greenhouse gas emissions and grapevine performance was evaluated and compared to a non-amended control using sensor-based analyses. Results: Increased CO2 emissions and lower N2O emissions were found for the incorporation treatments compared to the control, but these differences were not statistically significant due to high spatial variability. Only few plant traits like chlorophyll content or berry cuticle characteristics were significantly affected in some of the experimental years. Over the course of the study, annual climatic conditions had a much stronger influence on plant vigor and grape quality than the incorporated organic amendments. Discussion: In summary, organic soil amendments and their deep incorporation did not have any significant effect on greenhouse gas emissions and no measurable or only negligible effect on grapevine vigor, and grape quality parameters. Thus, according to our study the deposition of organic amendments in vineyard subsoils seems to be an option for viticulture to contribute to carbon storage in soils in order to mitigate climate change.

Cytopathic SARS-CoV-2 screening on VERO-E6 cells in a large-scale repurposing effort.

Worldwide, there are intensive efforts to identify repurposed drugs as potential therapies against SARS-CoV-2 infection and the associated COVID-19 disease. To date, the anti-inflammatory drug dexamethasone and (to a lesser extent) the RNA-polymerase inhibitor remdesivir have been shown to be effective in reducing mortality and patient time to recovery, respectively, in patients. Here, we report the results of a phenotypic screening campaign within an EU-funded project (H2020-EXSCALATE4COV) aimed at extending the repertoire of anti-COVID therapeutics through repurposing of available compounds and highlighting compounds with new mechanisms of action against viral infection. We screened 8702 molecules from different repurposing libraries, to reveal 110 compounds with an anti-cytopathic IC50 < 20 µM. From this group, 18 with a safety index greater than 2 are also marketed drugs, making them suitable for further study as potential therapies against COVID-19. Our result supports the idea that a systematic approach to repurposing is a valid strategy to accelerate the necessary drug discovery process.

Towards Sensor-Based Phenotyping of Physical Barriers of Grapes to Improve Resilience to Botrytis Bunch Rot.

Botrytis bunch rot is one of the economically most important fungal diseases in viticulture (aside from powdery mildew and downy mildew). So far, no active defense mechanisms and resistance loci against the necrotrophic pathogen are known. Since long, breeders are mostly selecting phenotypically for loose grape bunches, which is recently the most evident trait to decrease the infection risk of Botrytis bunch rot. This study focused on plant phenomics of multiple traits by applying fast sensor technologies to measure berry impedance (Z REL ), berry texture, and 3D bunch architecture. As references, microscopic determined cuticle thickness (MS CT ) and infestation of grapes with Botrytis bunch rot were used. Z REL hereby is correlated to grape bunch density OIV204 (r = -0.6), cuticle thickness of berries (r = 0.61), mean berry diameter (r = -0.63), and Botrytis bunch rot (r = -0.7). However, no correlation between Z REL and berry maturity or berry texture was observed. In comparison to the category of traditional varieties (mostly susceptible), elite breeding lines show an impressive increased Z REL value (+317) and a 1-µm thicker berry cuticle. Quantitative trait loci (QTLs) on LGs 2, 6, 11, 15, and 16 were identified for Z REL and berry texture explaining a phenotypic variance of between 3 and 10.9%. These QTLs providing a starting point for the development of molecular markers. Modeling of Z REL and berry texture to predict Botrytis bunch rot resilience revealed McFadden R 2 = 0.99. Taken together, this study shows that in addition to loose grape bunch architecture, berry diameter, Z REL , and berry texture values are probably additional parameters that could be used to identify and select Botrytis-resilient wine grape varieties. Furthermore, grapevine breeding will benefit from these reliable methodologies permitting high-throughput screening for additional resilience traits of mechanical and physical barriers to Botrytis bunch rot. The findings might also be applicable to table grapes and other fruit crops like tomato or blueberry.

Identification of Inhibitors of SARS-CoV-2 3CL-Pro Enzymatic Activity Using a Small Molecule in Vitro Repurposing Screen.

Compound repurposing is an important strategy for the identification of effective treatment options against SARS-CoV-2 infection and COVID-19 disease. In this regard, SARS-CoV-2 main protease (3CL-Pro), also termed M-Pro, is an attractive drug target as it plays a central role in viral replication by processing the viral polyproteins pp1a and pp1ab at multiple distinct cleavage sites. We here report the results of a repurposing program involving 8.7 K compounds containing marketed drugs, clinical and preclinical candidates, and small molecules regarded as safe in humans. We confirmed previously reported inhibitors of 3CL-Pro and have identified 62 additional compounds with IC50 values below 1 µM and profiled their selectivity toward chymotrypsin and 3CL-Pro from the Middle East respiratory syndrome virus. A subset of eight inhibitors showed anticytopathic effect in a Vero-E6 cell line, and the compounds thioguanosine and MG-132 were analyzed for their predicted binding characteristics to SARS-CoV-2 3CL-Pro. The X-ray crystal structure of the complex of myricetin and SARS-Cov-2 3CL-Pro was solved at a resolution of 1.77 Å, showing that myricetin is covalently bound to the catalytic Cys145 and therefore inhibiting its enzymatic activity.

EU-OPENSCREEN: A Novel Collaborative Approach to Facilitate Chemical Biology.

Compound screening in biological assays and subsequent optimization of hits is indispensable for the development of new molecular research tools and drug candidates. To facilitate such discoveries, the European Research Infrastructure EU-OPENSCREEN was founded recently with the support of its member countries and the European Commission. Its distributed character harnesses complementary knowledge, expertise, and instrumentation in the discipline of chemical biology from 20 European partners, and its open working model ensures that academia and industry can readily access EU-OPENSCREEN's compound collection, equipment, and generated data. To demonstrate the power of this collaborative approach, this perspective article highlights recent projects from EU-OPENSCREEN partner institutions. These studies yielded (1) 2-aminoquinazolin-4(3 H)-ones as potential lead structures for new antimalarial drugs, (2) a novel lipodepsipeptide specifically inducing apoptosis in cells deficient for the pVHL tumor suppressor, (3) small-molecule-based ROCK inhibitors that induce definitive endoderm formation and can potentially be used for regenerative medicine, (4) potential pharmacological chaperones for inborn errors of metabolism and a familiar form of acute myeloid leukemia (AML), and (5) novel tankyrase inhibitors that entered a lead-to-candidate program. Collectively, these findings highlight the benefits of small-molecule screening, the plethora of assay designs, and the close connection between screening and medicinal chemistry within EU-OPENSCREEN.

Polymorphic microsatellite sites in the PRNP region point to excess of homozygotes in Creutzfeldt-Jakob disease patients.

Polymorphic microsatellite sites within 148 kb of the human prion gene complex, including the genes PRNP, PRND and PRNT, were analysed together with the Codon129 variants regarding 50 CJD (Creutzfeldt-Jakob Disease) patients and 46 non-diseased control persons. Three of the sites (MM03, MM04, Codon129) differed significantly (P<0.05) for their allele frequencies between the two groups--the predominant allele being always more frequent in the CJD group. Deviations from Hardy-Weinberg Equilibrium were mainly obtained in the CJD group--in all cases with a reduction of the observed heterozygosity. The sites MM03, MM04 and Codon129 were also analysed for their haplotypes. The predominant homozygous haplotype combination was more frequently observed in the CJD group (0.875) than in the non-diseased group (0.38). Thus the different polymorphic sites indicate that high CJD disposition is associated with homozygosity in the PRNP gene.

Heat-shock-mediated elimination of the nptII marker gene in transgenic apple (Malus×domestica Borkh.).

Production of marker-free genetically modified (GM) plants is one of the major challenges of molecular fruit breeding. Employing clean vector technologies, allowing the removal of undesired DNA sequences from GM plants, this goal can be achieved. The present study describes the establishment of a clean vector system in apple Malus×domestica Borkh., which is based on the use of the neomycin phosphotransferase II gene (nptII) as selectable marker gene and kanamycin/paramomycin as selective agent. The nptII gene can be removed after selection of GM shoots via site-specific excision mediated by heat-shock-inducible expression of the budding yeast FLP recombinase driven by the soybean Gmhsp17.5-E promoter. We created a monitoring vector containing the nptII and the flp gene as a box flanked by two direct repeats of the flp recognition target (FRT) sites. The FRT-flanked box separates the gusA reporter gene from the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter. Consequently, GUS expression does only occur after elimination of the FRT-flanked box. Transformation experiments using the monitoring vector resulted in a total of nine transgenic lines. These lines were investigated for transgenicity by PCR, RT-PCR and Southern hybridization. Among different temperature regimes tested, exposure to 42 °C for 3.5 to 4h led to efficient induction of FLP-mediated recombination and removal of the nptII marker gene. A second round of shoot regeneration from leaf explants led to GM apple plants completely free of the nptII gene.