Identification of bromelain subfamily proteases encoded in the pineapple genome.

Papain (aka C1A) family proteases, including bromelain enzymes, are widespread across the plant kingdom and play critical regulatory functions in protein turnover during development. The proteolytic activity exhibited by papain family proteases has led to their increased usage for a wide range of cosmetic, therapeutic, and medicinal purposes. Bromelain enzymes, or bromelains in short, are members of the papain family that are specific to the bromeliad plant family. The only major commercial extraction source of bromelain is pineapple. The importance of C1A family and bromelain subfamily proteases in pineapple development and their increasing economic importance led several researchers to utilize available genomic resources to identify protease-encoding genes in the pineapple genome. To date, studies are lacking in screening bromelain genes for targeted use in applied science studies. In addition, the bromelain genes coding for the enzymes present in commercially available bromelain products have not been identified and their evolutionary origin has remained unclear. Here, using the newly developed MD2 v2 pineapple genome, we aimed to identify bromelain-encoding genes and elucidate their evolutionary origin. Orthologous and phylogenetic analyses of all papain-family proteases encoded in the pineapple genome revealed a single orthogroup (189) and phylogenetic clade (XIII) containing the bromelain subfamily. Duplication mode and synteny analyses provided insight into the origin and expansion of the bromelain subfamily in pineapple. Proteomic analysis identified four bromelain enzymes present in two commercially available bromelain products derived from pineapple stem, corresponding to products of four putative bromelain genes. Gene expression analysis using publicly available transcriptome data showed that 31 papain-family genes identified in this study were up-regulated in specific tissues, including stem, fruit, and floral tissues. Some of these genes had higher expression in earlier developmental stages of different tissues. Similar expression patterns were identified by RT-qPCR analysis with leaf, stem, and fruit. Our results provide a strong foundation for future applicable studies on bromelain, such as transgenic approaches to increase bromelain content in pineapple, development of bromelain-producing bioreactors, and studies that aim to determine the medicinal and/or therapeutic viability of individual bromelain enzymes.

In Vitro Assessment of Bio-Functional Properties from Lactiplantibacillus plantarum Strains.

In recent years, alongside the conventional screening procedures for the evaluation of probiotics for human usage, the pharmaceutical and food industries have encouraged scientific research towards the selection of new probiotic bacterial strains with particular functional features. Therefore, this study intended to explore novel functional properties of five Lactiplantibacillus plantarum strains isolated from bee bread. Specifically, antioxidant, antimicrobial and ß-glucosidase activities, exopolysaccharides (EPS) production and the ability to synthesize γ-aminobutyric acid (GABA) were evaluated. The results demonstrated that the investigated L. plantarum strains were effective in inhibiting the growth of some human opportunistic pathogens in vitro (Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, Enterococcus faecalis and Staphylococcus aureus). Moreover, the evaluation of antioxidant and ß-glucosidase activity and of EPS and GABA production, revealed a different behavior among the strains, testifying how these properties are strongly strain-dependent. This suggests that a careful selection within a given species is important in order to identify appropriate strains for specific biotechnological applications. The results highlighted that the five strains of L. plantarum are promising candidates for application as dietary supplements in the human diet and as microbial cultures in specific food productions.

Recent Advances in the Biocontrol of Nosemosis in Honey Bees (Apis mellifera L.).

Nosemosis is a disease triggered by the single-celled spore-forming fungi Nosema apis and Nosema ceranae, which can cause extensive colony losses in honey bees (Apis mellifera L.). Fumagillin is an effective antibiotic treatment to control nosemosis, but due to its toxicity, it is currently banned in many countries. Accordingly, in the beekeeping sector, there is a strong demand for alternative ecological methods that can be used for the prevention and therapeutic control of nosemosis in honey bee colonies. Numerous studies have shown that plant extracts, RNA interference (RNAi) and beneficial microbes could provide viable non-antibiotic alternatives. In this article, recent scientific advances in the biocontrol of nosemosis are summarized.

Physicochemical properties, degradation kinetics, and antioxidant capacity of aqueous anthocyanin-based extracts from purple carrots compared to synthetic and natural food colorants.

As a means to evaluate the potential of carrot anthocyanins as food colorants and nutraceutical agents, we investigated the physicochemical stability and antioxidant capacity of purple carrot extracts under different pH (2.5-7.0) and temperature (4-40 °C) conditions, in comparison to a commercial synthetic (E131) and a natural grape-based (GRP) colorant. During incubation, the colorants were weekly-monitored for various color parameters, concentration of anthocyanins and phenolics, and antioxidant capacity. Carrot colorants were more stable than GRP; and their thermal stability was equal (at 4 °C) or higher than that of E131 (at 25-40 °C). Carrot anthocyanins had lower degradation rate at low pH and temperature, with acylated anthocyanins (AA) being significantly more stable than non-acylated anthocyanins (NAA). Anthocyanins acylated with feruloyl and coumaroyl glycosides were the most stable carrot pigments. The higher stability of carrot colorants is likely due to their richness in AA and -to a lesser extent- copigmentation with other phenolics.

Combined Use of Molecular Markers and High-Resolution Melting (HRM) to Assess Chromosome Dosage in Potato Hybrids.

In plants, the most widely used cytological techniques to assess parental genome contributions are based on in situ hybridization (FISH and GISH), but they are time-consuming and need specific expertise and equipment. Recent advances in genomics and molecular biology have made PCR-based markers a straightforward, affordable technique for chromosome typing. Here, we describe the development of a molecular assay that uses single-copy conserved ortholog set II (COSII)-based single nucleotide polymorphisms (SNPs) and the high-resolution melting (HRM) technique to assess the chromosome dosage of interspecific hybrids between a Solanum phureja-S. tuberosum diploid (2n = 2x = 24) hybrid and its wild relative S. commersonii. Screening and analysis of 45 COSII marker sequences allowed S. commersonii-specific SNPs to be identified for all 12 chromosomes. Combining the HRM technique with the establishment of synthetic DNA hybrids, SNP markers were successfully used to predict the expected parental chromosome ratio of 5 interspecific triploid hybrids. These results demonstrate the ability of this strategy to distinguish diverged genomes from each other, and to estimate chromosome dosage. The method could potentially be applied to any species as a tool to assess paternal to maternal ratios in the framework of a breeding program or following transformation techniques.

The Solanum commersonii Genome Sequence Provides Insights into Adaptation to Stress Conditions and Genome Evolution of Wild Potato Relatives.

Here, we report the draft genome sequence of Solanum commersonii, which consists of ∼830 megabases with an N50 of 44,303 bp anchored to 12 chromosomes, using the potato (Solanum tuberosum) genome sequence as a reference. Compared with potato, S. commersonii shows a striking reduction in heterozygosity (1.5% versus 53 to 59%), and differences in genome sizes were mainly due to variations in intergenic sequence length. Gene annotation by ab initio prediction supported by RNA-seq data produced a catalog of 1703 predicted microRNAs, 18,882 long noncoding RNAs of which 20% are shown to target cold-responsive genes, and 39,290 protein-coding genes with a significant repertoire of nonredundant nucleotide binding site-encoding genes and 126 cold-related genes that are lacking in S. tuberosum. Phylogenetic analyses indicate that domesticated potato and S. commersonii lineages diverged ∼2.3 million years ago. Three duplication periods corresponding to genome enrichment for particular gene families related to response to salt stress, water transport, growth, and defense response were discovered. The draft genome sequence of S. commersonii substantially increases our understanding of the domesticated germplasm, facilitating translation of acquired knowledge into advances in crop stability in light of global climate and environmental changes.

Potato Tuber Blight Resistance Phenotypes Correlate with RB Transgene Transcript Levels in an Age-Dependent Manner.

Plants have evolved strategies and mechanisms to detect and respond to pathogen attack. Different organs of the same plant may be subjected to different environments (e.g., aboveground versus belowground) and pathogens with different lifestyles. Accordingly, plants commonly need to tailor defense strategies in an organ-specific manner. Phytophthora infestans, causal agent of potato late blight disease, infects both aboveground foliage and belowground tubers. We examined the efficacy of transgene RB (known for conferring foliar late blight resistance) in defending against tuber late blight disease. Our results indicate that the presence of the transgene has a positive yet only marginally significant effect on tuber disease resistance on average. However, a significant association between transgene transcript levels and tuber resistance was established for specific transformed lines in an age-dependent manner, with higher transcript levels indicating enhanced tuber resistance. Thus, RB has potential to function in both foliage and tuber to impart late blight resistance. Our data suggest that organ-specific resistance might result directly from transcriptional regulation of the resistance gene itself.

A DArT marker-based linkage map for wild potato Solanum bulbocastanum facilitates structural comparisons between Solanum A and B genomes.

BACKGROUND: Wild potato Solanum bulbocastanum is a rich source of genetic resistance against a variety of pathogens. It belongs to a taxonomic group of wild potato species sexually isolated from cultivated potato. Consistent with genetic isolation, previous studies suggested that the genome of S. bulbocastanum (B genome) is structurally distinct from that of cultivated potato (A genome). However, the genome architecture of the species remains largely uncharacterized. The current study employed Diversity Arrays Technology (DArT) to generate a linkage map for S. bulbocastanum and compare its genome architecture with those of potato and tomato. RESULTS: Two S. bulbocastanum parental linkage maps comprising 458 and 138 DArT markers were constructed. The integrated map comprises 401 non-redundant markers distributed across 12 linkage groups for a total length of 645 cM. Sequencing and alignment of DArT clones to reference physical maps from tomato and cultivated potato allowed direct comparison of marker orders between species. A total of nine genomic segments informative in comparative genomic studies were identified. Seven genome rearrangements correspond to previously-reported structural changes that have occurred since the speciation of tomato and potato. We also identified two S. bulbocastanum genomic regions that differ from cultivated potato, suggesting possible chromosome divergence between Solanum A and B genomes. CONCLUSIONS: The linkage map developed here is the first medium density map of S. bulbocastanum and will assist mapping of agronomical genes and QTLs. The structural comparison with potato and tomato physical maps is the first genome wide comparison between Solanum A and B genomes and establishes a foundation for further investigation of B genome-specific structural chromosome rearrangements.

Biochemical features of native red wines and genetic diversity of the corresponding grape varieties from Campania region.

Campania region has always been considered one of the most appreciated Italian districts for wine production. Wine distinctiveness arises from their native grapevines. To better define the chemical profile of Campania autochthonous red grape varieties, we analysed the phenolic composition of Aglianico di Taurasi, Aglianico del Vulture, Aglianico del Taburno, Piedirosso wines, and a minor native variety, Lingua di Femmina in comparison with Merlot and Cabernet Sauvignon, as reference cultivars. A genetic profiling was also carried out using microsatellite molecular markers with high polymorphic and unambiguous profiles. Principal component analysis applied to 72 wines based on the 18 biochemical parameters, explained 77.6% of the total variance and highlighted important biological entities providing insightful patterns. Moreover, comparison of SSR-based data with phenylpropanoid molecules exhibited a statistically significant correlation. Our approach might be reasonably adopted for future characterisations and traceability of grapevines and corresponding wines.

Molecular mapping of vernalization requirement and fertility restoration genes in carrot.

Carrot (Daucus carota L.) is a cool-season vegetable normally classified as a biennial species, requiring vernalization to induce flowering. Nevertheless, some cultivars adapted to warmer climates require less vernalization and can be classified as annual. Most modern carrot cultivars are hybrids which rely upon cytoplasmic male-sterility for commercial production. One major gene controlling floral initiation and several genes restoring male fertility have been reported but none have been mapped. The objective of the present work was to develop the first linkage map of carrot locating the genomic regions that control vernalization response and fertility restoration. Using an F(2) progeny, derived from the intercross between the annual cultivar 'Criolla INTA' and a petaloid male sterile biennial carrot evaluated over 2 years, both early flowering habit, which we name Vrn1, and restoration of petaloid cytoplasmic male sterility, which we name Rf1, were found to be dominant traits conditioned by single genes. On a map of 355 markers covering all 9 chromosomes with a total map length of 669 cM and an average marker-to-marker distance of 1.88 cM, Vrn1 mapped to chromosome 2 with flanking markers at 0.70 and 0.46 cM, and Rf1 mapped to chromosome 9 with flanking markers at 4.38 and 1.12 cM. These are the first two reproductive traits mapped in the carrot genome, and their map location and flanking markers provide valuable tools for studying traits important for carrot domestication and reproductive biology, as well as facilitating carrot breeding.

Higher copy numbers of the potato RB transgene correspond to enhanced transcript and late blight resistance levels.

Late blight of potato ranks among the costliest of crop diseases worldwide. Host resistance offers the best means for controlling late blight, but previously deployed single resistance genes have been short-lived in their effectiveness. The foliar blight resistance gene RB, previously cloned from the wild potato Solanum bulbocastanum, has proven effective in greenhouse tests of transgenic cultivated potato. In this study, we examined the effects of the RB transgene on foliar late blight resistance in transgenic cultivated potato under field production conditions. In a two-year replicated trial, the RB transgene, under the control of its endogenous promoter, provided effective disease resistance in various genetic backgrounds, including commercially prominent potato cultivars, without fungicides. RB copy numbers and transcript levels were estimated with transgene-specific assays. Disease resistance was enhanced as copy numbers and transcript levels increased. The RB gene, like many other disease resistance genes, is constitutively transcribed at low levels. Transgenic potato lines with an estimated 15 copies of the RB transgene maintain high RB transcript levels and were ranked among the most resistant of 57 lines tested. We conclude that even in these ultra-high copy number lines, innate RNA silencing mechanisms have not been fully activated. Our findings suggest resistance-gene transcript levels may have to surpass a threshold before triggering RNA silencing. Strategies for the deployment of RB are discussed in light of the current research.

Changes in disease resistance phenotypes associated with plant physiological age are not caused by variation in R gene transcript abundance.

Foliar late blight is one of the most important diseases of potato. Foliar blight resistance has been shown to change as a plant ages. In other pathosystems, resistance (R) gene transcript levels appear to be correlated to disease resistance. The cloning of the broad-spectrum, foliar blight resistance gene RB provided the opportunity to explore how foliar blight resistance and R-gene transcript levels vary with plant age. Plants of Solanum bulbocastanum PT29, from which RB, including the native promoter and other flanking regions, was cloned, and S. tuberosum cv. Dark Red Norland (nontransformed and RB-transformed) representing three different developmental stages were screened for resistance to late blight and RB transcript levels. Preflowering plants of all genotypes exhibited the highest levels of resistance, followed by postflowering and near-senescing plants. The RB transgene significantly affected resistance, enhancing resistance levels of all RB-containing lines, especially in younger plants. RB transgene transcripts were detected at all plant ages, despite weak correlation with disease resistance. Consistent transcript levels in plants of different physiological ages with variable levels of disease resistance demonstrate that changes in disease-resistance phenotypes associated with plant age cannot be attributed to changes in R-gene transcript abundance.