Molecular and catalytic characterization of the herbicide-inducible glutathione transferases from Phaseolus vulgaris

Plant glutathione transferases (GSTs) comprise a large family of inducible enzymes that play important roles in stress tolerance and herbicide detoxification. Treatment of Phaseolus vulgaris leaves with the aryloxyphenoxypropionic herbicide fluazifop-p-butyl resulted in induction of GST activities. Three inducible GST isoenzymes were identified and separated by affinity chromatography. Their full-length cDNAs with complete open reading frame were isolated using RACE-RT and information from N-terminal amino acid sequences. Analysis of the cDNA clones showed that the deduced amino acid sequences share high homology with GSTs that belong to phi and tau classes. The three isoenzymes were expressed in E. coli and their substrate specificity was determined towards 20 different substrates. The results showed that the fluazifop-inducible glutathione transferases from P. vulgaris (PvGSTs) catalyze a broad range of reactions and exhibit quite varied substrate specificity. Molecular modeling and structural analysis was used to identify key structural characteristics and to provide insights into the substrate specificity and the catalytic mechanism of these enzymes. These results provide new insights into catalytic and structural diversity of GSTs and the detoxifying mechanism used by P. vulgaris

Application of the ITS2 region for barcoding plants of the genus Triticum L. and Aegilops L.

Molecular taxonomic studies have been performed in the past in order to identify different wheat species and construct a molecular phylogeny. These were based on universal but sufficiently divergent sequences from both the nuclear and chloroplastic genomes of wheat. They included two short plastid sequences from the plastid genes rbcL and matK which have been proposed as the core “barcode” sequences by the “barcoding” guidelines for general plant identification. Historically, in molecular plant taxonomy, plastidic sequences had been favored over nuclear sequences, due to their uniparental inheritance and consequently lower intra-molecular recombination. However recently, the short nuclear sequence from the internal transcribed spacer 2 (ITS2) has been used successfully for the accurate identification of many medicinal and other plant species. Herein, we have used the plastidic matK, rbcL trnL, and the nuclear ITS2 region for the identification of different wheat species of Triticum L. and goatgrass species of Aegilops L. We have successfully discriminated all species that were examined from both genera, thus, validating the ITS2 region as a ‘barcode tool’ for accurate distinction of plants in the genus Triticum L. and Aegilops L. The success rate of PCR amplification and sequencing of the ITS2 region was 100%. We report also that matK, rbcL and trnL regions could not discriminate all species in contrast to the ITS2 region which demonstrated full discriminatory capacity

Rapid analysis for the identification of the seagrass Halophila ovalis (Hydrocharitaceae)

Seagrasses are considered as one of the most important species as they play key ecological roles in various types of ecosystems and also provide a food source for endangered animal species. There are two main  characteristics of seagrasses that hinder efforts to correctly identify species based on conventional identification keys alone: i) the variability of  morphological characteristics and ii) lack of needed morphological  characters especially flowers. A taxonomically unresolved complex such as Halophila spp. is reported. Plant DNA barcoding regions (rbcL and trnH-psbA) were used to confirm species of collected seagrasses from the southern coast of Thailand. Small and big-leaved samples of Halophila spp. were analysed in this study. The big-leaved samples were identified on the field as Halophila ovalis whilst it was uncertain whether the small-leaved samples belonged to H. ovalis. DNA analysis revealed that the small-leaved samples could be H. ovalis. We also coupled PCR with high resolution melt (HRM) to more cost-effectively identify individuals of H. ovilis than using barcoding alone. Using HRM to resolve differences in the sequence of two genes showed that the two unknown seagrasses belonged to the same species as H. ovalis.  In conclusion, using HRM proved to pose great potential in seagrass identification. Key words: DNA barcoding, Halophila ovalis, rbcL, trnH-psbA, species identification

Advances of DNA-based methods for tracing the botanical origin of food products

The need for accurate and reliable methods for plant species identification in nature and in food products has steadily increased during past decades, particularly with the recent food scares and the development of trade and technological progress in food production. Moreover, the development of high added value products based on plants has raised concerns about adulteration. Thus, reliable methods to protect the producer, the company and the customer are needed. Fresh food products without any processing are suitable for many types of analytical or molecular analyses. But as most of foodstuff samples are processed to some extent, DNA is usually altered and fragmented into small fragments. However, extensive research has been performed and DNA based methods for food authenticity are becoming the methods of choice. Herein DNA based methods for species identification and authenticity in foods as well as quantitation methods, are based on DNA. These methodologies progress extremely fast; thus a review on the current state of the art on DNA based methods is useful in order to assess the field. The problems, advantages and disadvantages of the methods are also discussed. The trend of high throughput DNA technologies is recognized

Genotyping of Listeria monocytogenes isolates from poultry carcasses using high resolution melting (HRM) analysis.

An outbreak situation of human listeriosis requires a fast and accurate protocol for typing Listeria monocytogenes. Existing techniques are either characterized by low discriminatory power or are laborious and require several days to give a final result. Polymerase chain reaction (PCR) coupled with high resolution melting (HRM) analysis was investigated in this study as an alternative tool for a rapid and precise genotyping of L. monocytogenes isolates. Fifty-five isolates of L. monocytogenes isolated from poultry carcasses and the environment of four slaughterhouses were typed by HRM analysis using two specific markers, internalin B and ssrA genes. The analysis of genotype confidence percentage of L. monocytogenes isolates produced by HRM analysis generated dendrograms with two major groups and several subgroups. Furthermore, the analysis of the HRM curves revealed that all L. monocytogenes isolates could easily be distinguished. In conclusion, HRM was proven to be a fast and powerful tool for genotyping isolates of L. monocytogenes

Advances of DNA-based methods for tracing the botanical origin of food products

The need for accurate and reliable methods for plant species identification in nature and in food products has steadily increased during past decades, particularly with the recent food scares and the development of trade and technological progress in food production. Moreover, the development of high added value products based on plants has raised concerns about adulteration. Thus, reliable methods to protect the producer, the company and the customer are needed. Fresh food products without any processing are suitable for many types of analytical or molecular analyses. But as most of foodstuff samples are processed to some extent, DNA is usually altered and fragmented into small fragments. However, extensive research has been performed and DNA based methods for food authenticity are becoming the methods of choice. Herein DNA based methods for species identification and authenticity in foods as well as quantitation methods, are based on DNA. These methodologies progress extremely fast; thus a review on the current state of the art on DNA based methods is useful in order to assess the field. The problems, advantages and disadvantages of the methods are also discussed. The trend of high throughput DNA technologies is recognized

Vegetable Grafting From a Molecular Point of View: The Involvement of Epigenetics in Rootstock-Scion Interactions

Vegetable grafting is extensively used today in agricultural production to control soil-borne pathogens, abiotic and biotic stresses and to improve phenotypic characteristics of the scion. Commercial vegetable grafting is currently practiced in tomato, watermelon, melon, eggplant, cucumber, and pepper. It is also regarded as a rapid alternative to the relatively slow approach of breeding for increased environmental-stress tolerance of fruit vegetables. However, even though grafting has been used for centuries, until today, there are still many issues that have not been elucidated. This review will emphasize on the important mechanisms taking place during grafting, especially the genomic interactions between grafting partners and the impact of rootstocks in scion’s performance. Special emphasis will be drawn on the relation between vegetable grafting, epigenetics, and the changes in morphology and quality of the products. Recent advances in plant science such as next-generation sequencing provide new information regarding the molecular interactions between rootstock and scion. It is now evidenced that genetic exchange is happening across grafting junctions between rootstock and scion, potentially affecting grafting-mediated effects already recorded in grafted plants. Furthermore, significant changes in DNA methylation are recorded in grafted scions, suggesting that these epigenetic mechanisms could be implicated in grafting effects. In this aspect, we also discuss the process and the molecular aspects of rootstock scion communication. Finally, we provide with an extensive overview of gene expression changes recorded in grafted plants and how these are related to the phenotypic changes observed. Τhis review finally seeks to elucidate the dynamics of rootstock-scion interactions and thus stimulate more research on grafting in the future. In a future where sustainable agricultural production is the way forward, grafting could play an important role to develop products of higher yield and quality in a safe and “green” way. © Copyright © 2021 Tsaballa, Xanthopoulou, Madesis, Tsaftaris and Nianiou-Obeidat

High-Resolution Melting approaches towards plant fungal molecular diagnostics (vol 43, pg 265, 2015)

Reliable and early molecular detection of phytopathogenic fungi is crucial in an era where the expansion of global trade in plant material is undoubtedly increasing the risk of invasive outbreaks, with devastating effects in crop production. Genetic variation within and between fungal species or strains is also important for screening isolates regarding various resistance attributes. Until today many approaches have been employed in fungal diagnostics which are either labor- and time-consuming or costly and of reduced sensitivity. Here, we demonstrate and review recent advances on high-resolution melting (HRM) analysis as a rapid, accurate and powerful tool, capable of differentiating even closely related fungal isolates. HRM technique is based on monitoring the melting of PCR amplicons, using saturating concentrations of a fluorescent intercalating dye that binds to double-stranded DNA. Additionally, we discuss the four case studies inferring applications of HRM analysis towards either genotyping of closely related fungal species or screening for fungicide resistance. We focus on the promising results of these studies, giving some technical considerations and describing the advantages of the application of this approach. Finally, we discuss current prospects and applications for research and development related to this innovative HRM technique in plant fungal diagnostics