Regulation of juvenility in Antirrhinum majus

Floral initiation is regulated by an elaborate network of signalling pathways, including the photoperiodic pathway. In Arabidopsis, flowering is promoted through this pathway by activation of FLOWERING LOCUS T (FT) by CONSTANS (CO) in long days. During juvenility plants are incapable of flowering in response to environmental conditions that would normally be favourable. This project studies the molecular basis of floral incompetence during juvenility in the model annual species, Antirrhinum majus and the important commercial tree species, Olea europaea, which has an extended juvenile phase. Photoperiod transfer experiments were used to measure the length of juvenility in plants grown in controlled environment cabinets at different Daily Light Integrals. Analysis of Antirrhinum FT (AmFT) expression during development showed that AmFT expression is minimal during juvenility and increases in all leaves following the end of the juvenile phase. The photoperiodic pathway was shown to be active during juvenility, suggesting that an additional mechanism involving the repression of FT could be involved in the regulation of juvenility. Full length Antirrhinum and Olive cDNAs representing homologues of the Arabidopsis FT repressors TEMPRANILLO 1 (AtTEM1) and AtTEM2, which act antagonistically with CO, were isolated. Molecular and phylogenetic analyses revealed high amino acid identities between Antirrhinum (AmTEM) and Olive (OeTEM) TEM-like proteins and AtTEM1 & 2. AmTEM and OeTEM proteins contain AP2 and B3 domains, consistent with AtTEM1 and AtTEM2, and can be classified as Class I members of the RAV sub-family of B3 transcription factors. AmTEM and OeTEM expression levels were shown to be higher during juvenility suggesting a potential role for TEM in controlling juvenility. A reciprocal relationship between expression levels of AmTEM/AtTEM1 and AmFT/AtFT was revealed in both Antirrhinum and Arabidopsis. Analysis of expression across development showed that AmTEM/AtTEM1 levels decline at around the time juvenility ends corresponding to when AmFT/AtFT levels start to increase. Arabidopsis tem1 mutants over-expressing AmTEM, OeTEM or AtTEM1 exhibited delayed flowering compared to the tem1 mutant, which demonstrated their role in regulating flowering time. Over-expression of AmTEM was shown to increase the length of the juvenile phase, delay the induction of AtCO and AtFT expression and reduce the overall levels of AtFT expression. Conversely, the juvenile phases of tem1 single and tem1/2 double mutants were shown to be shorter than in wild-type plants, with the induction of AtCO and AtFT expression occurring earlier. These findings are consistent with a role for TEM in regulating juvenility, which occurs through the down-regulation of FT and CO, and results in the inability to proceed to reproductive growth

Regulation of juvenility in Antirrhinum majus

Floral initiation is regulated by an elaborate network of signalling pathways, including the photoperiodic pathway. In Arabidopsis, flowering is promoted through this pathway by activation of FLOWERING LOCUS T (FT) by CONSTANS (CO) in long days. During juvenility plants are incapable of flowering in response to environmental conditions that would normally be favourable. This project studies the molecular basis of floral incompetence during juvenility in the model annual species, Antirrhinum majus and the important commercial tree species, Olea europaea, which has an extended juvenile phase. Photoperiod transfer experiments were used to measure the length of juvenility in plants grown in controlled environment cabinets at different Daily Light Integrals. Analysis of Antirrhinum FT (AmFT) expression during development showed that AmFT expression is minimal during juvenility and increases in all leaves following the end of the juvenile phase. The photoperiodic pathway was shown to be active during juvenility, suggesting that an additional mechanism involving the repression of FT could be involved in the regulation of juvenility. Full length Antirrhinum and Olive cDNAs representing homologues of the Arabidopsis FT repressors TEMPRANILLO 1 (AtTEM1) and AtTEM2, which act antagonistically with CO, were isolated. Molecular and phylogenetic analyses revealed high amino acid identities between Antirrhinum (AmTEM) and Olive (OeTEM) TEM-like proteins and AtTEM1 & 2. AmTEM and OeTEM proteins contain AP2 and B3 domains, consistent with AtTEM1 and AtTEM2, and can be classified as Class I members of the RAV sub-family of B3 transcription factors. AmTEM and OeTEM expression levels were shown to be higher during juvenility suggesting a potential role for TEM in controlling juvenility. A reciprocal relationship between expression levels of AmTEM/AtTEM1 and AmFT/AtFT was revealed in both Antirrhinum and Arabidopsis. Analysis of expression across development showed that AmTEM/AtTEM1 levels decline at around the time juvenility ends corresponding to when AmFT/AtFT levels start to increase. Arabidopsis tem1 mutants over-expressing AmTEM, OeTEM or AtTEM1 exhibited delayed flowering compared to the tem1 mutant, which demonstrated their role in regulating flowering time. Over-expression of AmTEM was shown to increase the length of the juvenile phase, delay the induction of AtCO and AtFT expression and reduce the overall levels of AtFT expression. Conversely, the juvenile phases of tem1 single and tem1/2 double mutants were shown to be shorter than in wild-type plants, with the induction of AtCO and AtFT expression occurring earlier. These findings are consistent with a role for TEM in regulating juvenility, which occurs through the down-regulation of FT and CO, and results in the inability to proceed to reproductive growth.EThOS - Electronic Theses Online ServiceUniversity of WarwickGBUnited Kingdo

Molecular Verification of the UK National Collection of Cultivated Liriope and Ophiopogon Plants

open access articleA collection of cultivated Liriope and Ophiopogon plants was established in 1996–1998 and subsequently hosted at a horticultural college. Uncertainties about the identification of the accessions, compounded by potential errors in propagation and labelling have led to waning confidence in the identities of the plants in the collection. The potential for using DNA barcoding to determine the species identities of the accessions was investigated. The DNA barcode regions of the plastid ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene (rbcL) and nuclear ribosomal internal transcribed spacer (nrITS) were amplified. DNA sequence analysis allowed the sequences of the accessions to be compared to reference sequences in public databases. A simple haplotype map of the characteristic polymorphic positions in the rbcL regions was used to clearly distinguish between the two genera and assign Ophiopogon accessions to individual species or sub-groups of species. The ITS sequence data confirmed these genus and species assignations and provided greater resolution to distinguish between closely related species. The combination of two DNA barcodes allowed most of the accessions to be assigned to individual species. This molecular verification confirmed the identity of about 70% of the accessions, with the remaining 30% demonstrating a range of mistaken identities at the species and genus level

DNA Authentication of St John’s Wort (Hypericum perforatum L.) Commercial Products Targeting the ITS Region

open access articleThere is considerable potential for the use of DNA barcoding methods to authenticate raw medicinal plant materials, but their application to testing commercial products has been controversial. A simple PCR test targeting species-specific sequences within the nuclear ribosomal internal transcribed spacer (ITS) region was adapted to screen commercial products for the presence of Hypericum perforatum L. material. DNA differing widely in amount and extent of fragmentation was detected in a number of product types. Two assays were designed to further analyse this DNA using a curated database of selected Hypericum ITS sequences: A qPCR assay based on a species-specific primer pair spanning the ITS1 and ITS2 regions, using synthetic DNA reference standards for DNA quantitation and a Next Generation Sequencing (NGS) assay separately targeting the ITS1 and ITS2 regions. The ability of the assays to detect H. perforatum DNA sequences in processed medicines was investigated. Out of twenty different matrices tested, both assays detected H. perforatum DNA in five samples with more than 103 ITS copies µL−1 DNA extract, whilst the qPCR assay was also able to detect lower levels of DNA in two further samples. The NGS assay confirmed that H. perforatum was the major species in all five positive samples, though trace contaminants were also detected

Selection of reference genes for diurnal and developmental time-course real-time PCR expression analyses in lettuce

Background: Real-time quantitative polymerase chain reaction (RT-qPCR) analysis is a low cost and sensitive technique that is widely used to measure levels of gene expression. Selecting and validating appropriate reference genes for normalising target gene expression should be the first step in any expression study to avoid inaccurate results. Results: In this study, ten candidate genes were tested for their suitability for use as reference genes in diurnal and developmental timecourse experiments in lettuce. The candidate reference genes were then used to normalise the expression pattern of the FLOWERING LOCUS T (FT) gene, one of key genes involved in the flowering time pathway whose expression is known to vary throughout the day and at different stages of development. Three reference genes, LsPP2A-1 (PROTEIN PHOSPHATASE 2A-1), LsPP2AA3 (PROTEIN PHOSPHATASE 2A REGULATORY SUBUNIT A3) and LsTIP41 (TAP42-INTERACTING PROTEIN OF 41 kDa), were the most stably expressed candidate reference genes throughout both the diurnal and developmental timecourse experiments. In the developmental experiment using just LsPP2A-1 and LsTIP41 as reference genes would be sufficient for accurate normalisation, whilst in the diurnal experiment all three reference genes, LsPP2A-1, LsPP2AA3 and LsTIP41, would be necessary. The FT expression pattern obtained demonstrates that the use of multiple and robust reference genes for RT-qPCR expression analyses results in a more accurate and reliable expression profile. Conclusions: Reference genes suitable for use in diurnal and developmental timecourse experiments in lettuce were identified and used to produce a more accurate and reliable analysis of lsFT expression levels than previously obtained in such timecourse experiments

Genus-Specific Real-Time PCR and HRM Assays to Distinguish Liriope from Ophiopogon Samples

open access articleLiriope and Ophiopogon species have a long history of use as traditional medicines across East Asia. They have also become widely used around the world for ornamental and landscaping purposes. The morphological similarities between Liriope and Ophiopogon taxa have made the taxonomy of the two genera problematic and caused confusion about the identification of individual specimens. Molecular approaches could be a useful tool for the discrimination of these two genera in combination with traditional methods. Seventy-five Liriope and Ophiopogon samples from the UK National Plant Collections of Ophiopogon and Liriope were analyzed. The 5′ end of the DNA barcode region of the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcLa) was used for the discrimination of the two genera. A single nucleotide polymorphism (SNP) between the two genera allowed the development of discriminatory tests for genus-level identification based on specific PCR and high-resolution melt curve (HRM) assays. The study highlights the advantage of incorporating DNA barcoding methods into plant identification protocols and provides simple assays that could be used for the quality assurance of commercially traded plants and herbal drugs

Challenges in Medicinal and Aromatic Plants DNA Barcoding—Lessons from the Lamiaceae

open access articleThe potential value of DNA barcoding for the identification of medicinal plants and authentication of traded plant materials has been widely recognized; however, a number of challenges remain before DNA methods are fully accepted as an essential quality control method by industry and regulatory authorities. The successes and limitations of conventional DNA barcoding are considered in relation to important members of the Lamiaceae. The mint family (Lamiaceae) contains over one thousand species recorded as having a medicinal use, with many more exploited in food and cosmetics for their aromatic properties. The family is characterized by a diversity of secondary products, most notably the essential oils (EOs) produced in external glandular structures on the aerial parts of the plant that typify well-known plants of the basil (Ocimum), lavender (Lavandula), mint (Mentha), thyme (Thymus), sage (Salvia) and related genera. This complex, species-rich family includes widely cultivated commercial hybrids and endangered wild-harvested traditional medicines, and examples of potential toxic adulterants within the family are explored in detail. The opportunities provided by next generation sequencing technologies to whole plastome barcoding and nuclear genome sequencing are also discussed with relevant examples

Biomimetic hydroxyapatite nanocrystals are an active carrier for Salmonella bacteriophages

open access articlePurpose: The use of bacteriophages represents a valid alternative to conventional antimicrobial treatments, overcoming the widespread bacterial antibiotic resistance phenomenon. In this work, we evaluated whether biomimetic hydroxyapatite (HA) nanocrystals are able to enhance some properties of bacteriophages. The final goal of this study was to demonstrate that biomimetic HA nanocrystals can be used for bacteriophage delivery in the context of bacterial infections, and contribute – at the same time – to enhance some of the biological properties of the same bacteriophages such as stability, preservation, antimicrobial activity, and so on. Materials and methods: Phage isolation and characterization were carried out by using Mitomycin C and following double-layer agar technique. The biomimetic HA water suspension was synthesized in order to obtain nanocrystals with plate-like morphology and nanometric dimensions. The interaction of phages with the HA was investigated by dynamic light scattering and Zeta potential analyses. The cytotoxicity and intracellular killing activities of the phage–HA complex were evaluated in human hepatocellular carcinoma HepG2 cells. The bacterial inhibition capacity of the complex was assessed on chicken minced meat samples infected with Salmonella Rissen. Results: Our data highlighted that the biomimetic HA nanocrystal–bacteriophage complex was more stable and more effective than phages alone in all tested experimental conditions. Conclusion: Our results evidenced the important contribution of biomimetic HA nanocrystals: they act as an excellent carrier for bacteriophage delivery and enhance its biological characteristics. This study confirmed the significant role of the mineral HA when it is complexed with biological entities like bacteriophages, as it has been shown for molecules such as lactoferrin

Sequence-Specific Detection of Aristolochia DNA – A Simple Test for Contamination of Herbal Products

open access articleHerbal medicines are used globally for their health benefits as an alternative therapy method to modern medicines. The market for herbal products has increased rapidly over the last few decades, but this has in turn increased the opportunities for malpractices such as contamination or substitution of products with alternative plant species. In the 1990s, a series of severe renal disease cases were reported in Belgium associated with weight loss treatment, in which the active species Stephania tetrandra was found to be substituted with Aristolochia fangchi. A. fangchi contains toxic aristolochic acids, which have been linked to kidney failure, as well as cancers of the urinary tract. Because of these known toxicities, herbal medicines containing these compounds, or potentially contaminated by these plants, have been restricted or banned in some countries, but they are still available via the internet and in alternate formulations. In this study, a DNA based method based on quantitative real-time PCR (qPCR) was tested to detect and distinguish Aristolochia subg. Siphisia (Duch.) O.C.Schmidt species from a range of medicinal plants that could potentially be contaminated with Aristolochia material. Specific primers were designed to confirm that Aristolochia subg. Siphisia can be detected, even in small amounts, if it is present in the products, fulfilling the aim of offering a simple, cheaper and faster solution than the chemical methods. A synthetic gBlock template containing the primer sequences was used as a reference standard to calibrate the qPCR assay and to estimate the copy number of a target gene per sample. Generic primers covering the conserved 5.8S rRNA coding region were used as internal control to verify DNA quality and also as a reference gene for relative quantitation. To cope with potentially degraded DNA, all qPCR primer sets were designed to generate PCR products of under 100 bp allowing detection and quantification of A. fangchi gBlock even when mixed with S. tetrandra gBlock in different ratios. All proportions of Aristolochia, from 100 to 2%, were detected. Using standards, associating the copy number to each start quantity, the detection limit was calculated and set to about 50 copies

Monochromic Radiations Provided by Light Emitted Diode (LED) Modulate Infection and Defense Response to Fire Blight in Pear Trees

open access articlePathogenesis-related (PR) proteins are part of the systemic signaling network that perceives pathogens and activates defenses in the plant. Eukaryotic and bacterial species have a 24-h ‘body clock’ known as the circadian rhythm. This rhythm regulates an organism’s life, modulating the activity of the phytochromes (phys) and cryptochromes (crys) and the accumulation of the corresponding mRNAs, which results in the synchronization of the internal clock and works as zeitgeber molecules. Salicylic acid accumulation is also under light control and upregulates the PR genes expression, increasing plants’ resistance to pathogens. Erwinia amylovora causes fire blight disease in pear trees. In this work, four bacterial transcripts (erw1-4), expressed in asymptomatic E. amylovora-infected pear plantlets, were isolated. The research aimed to understand how the circadian clock, light quality, and related photoreceptors regulate PR and erw genes expression using transgenic pear lines overexpressing PHYB and CRY1 as a model system. Plantlets were exposed to different circadian conditions, and continuous monochromic radiations (Blue, Red, and Far-Red) were provided by light-emitting diodes (LED). Results showed a circadian oscillation of PR10 gene expression, while PR1 was expressed without clear evidence of circadian regulation. Bacterial growth was regulated by monochromatic light: the growth of bacteria exposed to Far-Red did not differ from that detected in darkness; instead, it was mildly stimulated under Red, while it was significantly inhibited under Blue. In this regulatory framework, the active form of phytochrome enhances the expression of PR1 five to 15 fold. An ultradian rhythm was observed fitting the zeitgeber role played by CRY1. These results also highlight a regulating role of photoreceptors on the expression of PRs genes in non-infected and infected plantlets, which influenced the expression of erw genes. Data are discussed concerning the regulatory role of photoreceptors during photoperiod and pathogen attacks