Time indices of multiphasic development in genotypes of sweet cherry are similar from dormancy to cessation of pit growth.

BACKGROUND AND AIMS: The archetypical double sigmoid-shaped growth curve of the sweet cherry drupe (Prunus avium) does not address critical development from eco-dormancy to anthesis and has not been correlated to reproductive bud development. Accurate representation of the growth and development of post-anthesis ovaries is confounded by anthesis timing, fruiting-density and the presence of unfertilized and defective ovaries whose growth differs from those that persist to maturation. These factors were addressed to assess pre-anthesis and full-season growth and development of three sweet cherry cultivars, 'Chelan', 'Bing' and 'Sweetheart', differing primarily in seasonal duration and fruit size. METHODS: Volume was calculated from photographic measurements of reproductive buds, ovaries and pits at all phases of development. A population of unfertilized ovaries was produced using bee-exclusion netting to enable a statistical comparison with an open pollinated population to detect differences in size and shape between successful and failing fruit growth. Anthesis timing and fruiting-density were manipulated by floral extinction at the spur and whole-tree scales. Developmental time indices were analysed using polynomial curve fitting of log-transformed data supported by Richards and logistic functions of asymptotic growth of the pit and maturing fruit, respectively. KEY RESULTS: Pre-anthesis growth began at the completion of eco-dormancy. A slight decline in relative growth rate (RGR) was observed during bud scale separation approx. -16 d from anthesis (DFA) before resumption of exponential growth to a maximum about 14 DFA. After anthesis, reduced growth of unfertilized or defective ovaries was partly discriminated from successful fruit at 5 DFA and completely at 25 DFA. Time indices of RGR inflections were similar among cultivars when adjusted for anthesis date alone, until the end of pit growth. Asymptotic growth of the pit underpinned the declining growth rate of fruit at the end of the first exponential growth phase. Duration of the subsequent expansive growth phase accounted for genotypic differences in seasonal duration and final size. Pit size and final fruit size were inversely related to fruiting-density. CONCLUSIONS: Developmental differences among early, mid and late maturing cultivars were not detected until the final growth period.

A Concise Enantioselective Synthesis of Fluorinated Pyrazolo-Piperidine GSK3901383A Enabled by an Organocatalytic Aza-Michael Addition.

A highly enantioselective organocatalytic aza-Michael addition of 4-nitro-pyrazole to ethyl (E)-2,2-difluoro-5-oxopent-3-enoate has been developed. This reaction enabled a concise, four-step, stereoselective synthesis of highly functionalized 3,3-difluoro-4-pyrazolo-piperidine GSK3901383A, a key intermediate for the synthesis of a leucine-rich repeat kinase 2 inhibitor API. Computational analysis provided insight into the steric requirements of the catalytic system, enabling rational selection of a highly selective catalyst.

Investigation of the Formaldehyde-Catalyzed NNitrosation of Dialkyl Amines: An Automated Experimental and Kinetic Modelling Study Using Dibutylamine.

The potential for drug substances and drug products to contain low levels of N-nitrosamines is of continued interest to the pharmaceutical industry and regulatory authorities. Acid-promoted nitrosation mechanisms in solution have been investigated widely in the literature and are supported by kinetic modelling studies. Carbonyl compounds, particularly formaldehyde, which may be present as impurities in excipients and drug product packaging components or introduced during drug substance manufacturing processes are also known to catalyze nitrosation, but their impact on the risk of N-nitrosamine formation has not been systematically investigated to date. In this study, we experimentally investigated the multivariate impact of formaldehyde, nitrite and pH on N-nitrosation in aqueous solution using dibutylamine as a model amine. We augmented a published kinetic model by adding formaldehyde-catalyzed nitrosation reactions. We validated the new kinetic model vs. the experimental data and then used the model to systematically investigate the impact of formaldehyde levels on N-nitrosamine formation. Simulations of aqueous solution systems show that at low formaldehyde levels the formaldehyde-catalyzed mechanisms are insignificant in comparison to other routes. However, formaldehyde-catalyzed mechanisms can become more significant at neutral and high pH under higher formaldehyde levels. Model-based sensitivity analysis demonstrated that under high nitrite levels and low formaldehyde levels (where the rate of formaldehyde-catalyzed nitrosation is low compared to the acid-promoted pathways) the model can be used with kinetic parameters for model amines in the literature without performing additional experiments to fit amine-specific parameters. For other combinations of reaction parameters containing formaldehyde, the formaldehyde-catalyzed kinetics are non-negligible, and thus it is advised that, under such conditions, additional experiments should be conducted to reliably use the model.

Oxygenation alleviates waterlogging-caused damages to cherry rootstocks.

Waterlogging has occurred more frequently in recent years due to climate change, so it is a huge threat to crop yield and quality. Sweet cherry, a fruit tree with a high economic value, is sensitive to waterlogging stress. One of the most effective methods for enhancing the waterlogging tolerance of sweet cherries is to select waterlogging-tolerant rootstocks. However, the waterlogging tolerance of different cherry rootstocks, and the underlying mechanism remains uncharacterized. Thus, we first evaluated the waterlogging resistance of five sweet cherry rootstocks planted in China. The data showed that 'Gisela 12' and 'Colt' were the most waterlogging-sensitive and -tolerant among the five tested varieties, respectively. Oxygenation effectively alleviated the adverse impacts of waterlogging stress on cherry rootstocks. Moreover, we found that the waterlogging group had lower relative water content, Fv/Fm value, net photosynthetic rate, and higher antioxidant enzyme activities, whereas the oxygenated group performed better in all these parameters. RNA-Seq analysis revealed that numerous DEGs were involved in energy production, antioxidant metabolism, hormone metabolism pathways, and stress-related transcription factors. These findings will help provide management strategies to enhance the waterlogging tolerance of cherry rootstocks and thereby achieve higher yield and better quality of cherries.

Rapid gene-based SNP and haplotype marker development in non-model eukaryotes using 3'UTR sequencing.

BACKGROUND: Sweet cherry (Prunus avium L.), a non-model crop with narrow genetic diversity, is an important member of sub-family Amygdoloideae within Rosaceae. Compared to other important members like peach and apple, sweet cherry lacks in genetic and genomic information, impeding understanding of important biological processes and development of efficient breeding approaches. Availability of single nucleotide polymorphism (SNP)-based molecular markers can greatly benefit breeding efforts in such non-model species. RNA-seq approaches employing second generation sequencing platforms offer a unique avenue to rapidly identify gene-based SNPs. Additionally, haplotype markers can be rapidly generated from transcript-based SNPs since they have been found to be extremely utile in identification of genetic variants related to health, disease and response to environment as highlighted by the human HapMap project. RESULTS: RNA-seq was performed on two sweet cherry cultivars, Bing and Rainier using a 3' untranslated region (UTR) sequencing method yielding 43,396 assembled contigs. In order to test our approach of rapid identification of SNPs without any reference genome information, over 25% (10,100) of the contigs were screened for the SNPs. A total of 207 contigs from this set were identified to contain high quality SNPs. A set of 223 primer pairs were designed to amplify SNP containing regions from these contigs and high resolution melting (HRM) analysis was performed with eight important parental sweet cherry cultivars. Six of the parent cultivars were distantly related to Bing and Rainier, the cultivars used for initial SNP discovery. Further, HRM analysis was also performed on 13 seedlings derived from a cross between two of the parents. Our analysis resulted in the identification of 84 (38.7%) primer sets that demonstrated variation among the tested germplasm. Reassembly of the raw 3'UTR sequences using upgraded transcriptome assembly software yielded 34,620 contigs containing 2243 putative SNPs in 887 contigs after stringent filtering. Contigs with multiple SNPs were visually parsed to identify 685 putative haplotypes at 335 loci in 301 contigs. CONCLUSIONS: This approach, which leverages the advantages of RNA-seq approaches, enabled rapid generation of gene-linked SNP and haplotype markers. The general approach presented in this study can be easily applied to other non-model eukaryotes irrespective of the ploidy level to identify gene-linked polymorphisms that are expected to facilitate efficient Gene Assisted Breeding (GAB), genotyping and population genetics studies. The identified SNP haplotypes reveal some of the allelic differences in the two sweet cherry cultivars analyzed. The identification of these SNP and haplotype markers is expected to significantly improve the genomic resources for sweet cherry and facilitate efficient GAB in this non-model crop.

Internal freezing and heat loss of apple (Malus domestica Borkh.) and sweet cherry (Prunus avium L.) reproductive buds are decreased with cellulose nanocrystal dispersions.

Cold damage has caused more economic losses to fruit crop growers in the U.S. than any other weather hazard, making it a perennial concern for producers. Cellulose nanocrystals (CNCs) represent a new generation of renewable bio-nanomaterials, with many unique physical and chemical properties, including their low thermal conductivity. Our team has developed a process for creating CNC dispersions that can be sprayed onto woody perennial crops, forming a thin insulating film around buds which has been shown to increase cold tolerance. Using digital scanning calorimetry (DSC) on dormant apple (Malus domestica Borkh.) reproductive buds, we investigated the thermodynamic properties of plant materials treated with CNC dispersion at lower temperatures. Scanning electron microscopy (SEM) was used to evaluate the thickness of the CNC films and their deposition on the sweet cherry bud surface. Apple buds treated with 3% CNC exhibited lethal freezing at temperatures 3.2°C and 5.5°C lower than the untreated control when sampled 1 and 3 days after application, respectively. Additionally, the latent heat capacity (J/g) of the 3% CNC-treated buds was 46% higher compared with untreated buds 1 day after application, and this difference increased 3 days after application to 168% higher. The emissivity of cherry buds treated with 3% CNC was reduced by an average of 16% compared with the untreated buds. SEM was able to detect the dried films on the surface of the buds 3 days after application. Film thickness measured with SEM increased with material concentration. The emissivity, HTE, and LTE results show that CNC-treated reproductive buds released thermal energy at a slower rate than the untreated buds and, consequently, exhibited internal ice nucleation events at temperatures as much as 5.5°C lower. The increased enthalpy during the LTE in the CNC-treated apple buds shows more energy released at lethal internal freezing, indicating that CNC coatings are increasing the amount of supercooled water. The effects of CNC shown during the DSC tests were increased by CNC concentration and time post-application. These results suggest that CNC dispersions dry into nanofilms on the bud surface, which affects their thermodynamic processes at low temperatures.

Draft genome data of Prunus avium cv 'Stella'.

Prunus avium cv. 'Stella' total cellular DNA was isolated from emerging leaf tissue and sequenced using Roche 454 GS FLX Titanium, and Illumina HiSeq 2000 High Throughput Sequencing (HTS) technologies. Sequence data were filtered and trimmed to retain nucleotides corresponding to Phred score 30, and assembled with CLC Genomics Workbench v.6.0.1. A total of 107,531 contigs were assembled with 185 scaffolds with a maximum length of 132,753 nucleotides and an N50 value of 4,601. The average depth of coverage was 135.87 nucleotides with a median depth of coverage equal to 31.50 nucleotides. The draft 'Stella' genome presented here covers 77.8% of the estimated 352.9Mb P. avium genome and is expected to facilitate genetics and genomics research focused on identifying genes and quantitative trait loci (QTL) underlying important agronomic and consumer traits.

Transcriptome Analysis Reveals Potential Mechanisms for Ethylene-Inducible Pedicel-Fruit Abscission Zone Activation in Non-Climacteric Sweet Cherry (Prunus avium L.).

The harvesting of sweet cherry (Prunus avium L.) fruit is a labor-intensive process. The mechanical harvesting of sweet cherry fruit is feasible; however, it is dependent on the formation of an abscission zone at the fruit-pedicel junction. The natural propensity for pedicel-fruit abscission zone (PFAZ) activation varies by cultivar, and the general molecular basis for PFAZ activation is not well characterized. In this study, ethylene-inducible change in pedicel fruit retention force (PFRF) was recorded in a developmental time-course with a concomitant analysis of the PFAZ transcriptome from three sweet cherry cultivars. In 'Skeena', mean PFRF for both control and treatment fruit dropped below the 0.40 kg-force (3.92 N) threshold for mechanical harvesting, indicating the activation of a discrete PFAZ. In 'Bing', mean PFRF for both control and treatment groups decreased over time. However, a mean PFRF conducive to mechanical harvesting was achieved only in the ethylene-treated fruit. While in 'Chelan' the mean PFRF of the control and treatment groups did not meet the threshold required for efficient mechanical harvesting. Transcriptome analysis of the PFAZ region followed by the functional annotation, differential expression analysis, and gene ontology (GO) enrichment analyses of the data facilitated the identification of phytohormone-responsive and abscission-related transcripts, as well as processes that exhibited differential expression and enrichment in a cultivar-dependent manner over the developmental time-course. Additionally, read alignment-based variant calling revealed several short variants in differentially expressed genes, associated with enriched gene ontologies and associated metabolic processes, lending potential insight into the genetic basis for different abscission responses between the cultivars. These results provide genetic targets for the induction or inhibition of PFAZ activation, depending on the desire to harvest the fruit with or without the stem attached. Understanding the genetic mechanisms underlying the development of the PFAZ will inform future cultivar development while laying a foundation for mechanized sweet cherry harvest.

Rapid discovery and structure-activity profiling of novel inhibitors of human immunodeficiency virus type 1 protease enabled by the copper(I)-catalyzed synthesis of 1,2,3-triazoles and their further functionalization.

Building from the results of a computational screen of a range of triazole-containing compounds for binding efficiency to human immunodeficiency virus type 1 protease (HIV-1-Pr), a novel series of potent inhibitors has been developed. The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), which provides ready access to 1,4-disubstituted-1,2,3-triazoles, was used to unite a focused library of azide-containing fragments with a diverse array of functionalized alkyne-containing building blocks. In combination with direct screening of the crude reaction products, this method led to the rapid identification of a lead structure and readily enabled optimization of both azide and alkyne fragments. Replacement of the triazole with a range of alternative linkers led to greatly reduced protease inhibition; however, further functionalization of the triazoles at the 5-position gave a series of compounds with increased activity, exhibiting Ki values as low as 8 nM.

Development of a redox-free Mitsunobu reaction exploiting phosphine oxides as precursors to dioxyphosphoranes.

The development of the first redox-free protocol for the Mitsunobu reaction is described. This has been achieved by exploiting triphenylphosphine oxide--the unwanted by-product in the conventional Mitsunobu reaction--as the precursor to the active P(V) coupling reagent. Multinuclear NMR studies are consistent with hydroxyl activation via an alkoxyphosphonium salt.

Assignation of sweet cherry selections to 3 taste groupings based on perceived sweetness and sourness.

UNLABELLED: Providing consumers with basic taste properties of sweet cherries at point of purchase would allow consumers to make purchase decisions based on fruit's intrinsic sensory attributes. The objective of this study was to develop a model to predict taste-grouping assignation of cherries into the following categories: (1) low sweetness/high sourness, (2) balance between sweetness and sourness, and (3) high sweetness/low sourness. A sensory panel (n = 10) was trained to recognize sweetness and sourness in 5 cultivars of sweet cherries and assign a taste grouping based on the perceived balance of sweetness and sourness. Four of these same cultivars were then evaluated for sweetness and sourness by a consumer panel (n = 117) and instrumentally for titratable acidity (TA) and soluble solids concentration (SSC). Results showed that for 3 of the 4 cherry cultivars, the sweetness/sourness balance of the cherries was not significantly different as evaluated instrumentally or by the trained panel. However, the balance determined by the consumer and the trained panel was different for 3 of the 4 cherry cultivars (P < 0.05). Based on trained panel perceived sweetness and sourness, a multinomial logit model was developed to predict the assignation of cherry taste grouping. The likelihood of group assignment depended on both the perceived sweetness and sourness of the cherry, with taste groupings agreed upon for 3 of 5 sweet cherry cultivars. As previous studies have indicated a positive relationship between cherry sweetness and sourness to consumer acceptance, these groupings show promise for assisting consumers in cherry selection at the point of purchase. PRACTICAL APPLICATIONS: The prediction models proposed in this study suggest that both sweetness and sourness are important in the cherry characterization and the ratio between the 2 attributes may be appropriate for making taste-grouping assignments. These groupings may then be used to provide additional sensory information to consumers to assist them in cherry selection at the point of purchase.

Chirality transfer in the aza-[2,3]-Wittig sigmatropic rearrangement.

The aza-[2,3]-Wittig sigmatropic rearrangements of substrates derived from enantiomerically pure alanine, valine and serine with phenyl and ester anion stabilising groups were investigated for their efficiency in chirality transfer. It was found that a methyl substituent at the stereogenic centre of the rearrangement precursors was inadequate to control the alkene stereoselectivity and enantioselectivity of the rearrangement. Ester stabilised anions of valine and serine derivatives were the most successful with up to 66% yield, 14 : 1 alkene (E)-stereoselection and 88% chirality transfer. A limitation to the steric bulk of the stereogenic centre was noted in that the substituent has to be bulky enough to dictate alkene stereoselection, but not too large to compromise the directing effect of the activating phenyldimethyl silyl substituent on the anion stabilising group. Experimental evidence suggested a possible complimentary coordinating effect of an O-MOM serine substituent, which may assist alkene stereoselectivity and enantioselectivity.

Total synthesis of (+/-)-kainic Acid with an aza-[2,3]-Wittig sigmatropic rearrangement as the key stereochemical determining step.

A flexible route to the kainoid skeleton is exemplified by the synthesis of (+/-)-kainic acid from 3-butyn-1-ol. The route relies on the aza-[2,3]-Wittig sigmatropic rearrangement to efficiently install the relative stereochemistry between C2-C3. The C4 stereocenter was derived from a diastereocontrolled iodolactonization. The aza-[2,3]-Wittig rearrangement potentially allows structural diversity at C3 and the displacement of the tosyloxy group with retention of stereochemistry allows structural diversity at C4. The trans-C2 carboxylic acid functional group was found to be the most important for retention of stereochemistry at C4 upon treatment with a higher order cyano cuprate reagent.