Research interests of Horticultural Genetics & Biotechnology
The laboratory of Plant Biotechnology & GMO Testing serves a dual role:
- Hosts the Department of Horticultural Genetics & Biotechnology Research Personnel and post-graduate students which focus on the following research projects.
- Provides laboratory services to the industry on qualitative and quantitative GMO testing in agrofood products, animal feed, fish food, olive oil and their products and any other product made of or consist of Soya, maize, cotton etc
THE FUNCTIONAL ROLE OF PROLYL-4 HYDROXYLASES IN PLANT GROWTH AND DEVELOPMENT
The nature of cell wall proteins is as varied as the many functions of plant cell walls. They are usually rich in one or two amino acids, contain highly repetitive sequence domains, and are highly or poorly glycosylated. Among these are the hydroxyproline-rich glycoproteins (HRGPs) or extensins, the arabinogalactan proteins, the glycine-rich proteins (GRPs), the proline-rich proteins (PRPs), and chimeric proteins that contain extensins-like domains.
Prolyl 4-hydroxylases (P4Hs), the enzymes responsible for the posttranslational hydroxylation of peptide proline in cell wall proteins, has been well described in animals but has been little studied in plants. There is no available information at the molecular level and particularly on their expression patterns in plant growth and development. To this direction we currently study:
- The expression of Arabidopsis P4Hs in response to various stresses such as hypoxia, anoxia, wounding and heat stress
- Arabidopsis P4H T-DNA knock out mutants for interesting phenotypes
- Arabidopsis plants transformed with P4H RNAi constructs
A construct prepared by cloning the promoter of Atp4h7 into the GUS reporter plasmid pBI101 (Invitrogen) was used for the Agrobacterium-mediated transformation of Arabidopsis thaliana (Columbia) plants. Exposure of these plants to 3 hours of anoxia resulted in expression of GUS gene in the shoots and roots of the transformed plants, visible as a blue colour on incubation with the chromogenic GUS substrate X-Gal. No GUS activity was detected in transformed plants maintained in air.
Expression of Arabidopsis P4H genes under hypoxia. Real time RT-PCR analysis was undertaken using a series of cDNA samples prepared from the roots and shoots of Arabidopsis plants exposed to a hypoxia time course. Specific primer combinations derived from unique sequences within the genes encoding Arabidopsis P4H homologous proteins AtP4H1 (At2g43080), AtP4H2 (At2g17720), AtP4H4 (At5g18900), AtP4H5 (At3g06300), AtP4H7 (At3g28480) and AtP4H9 (At4g33910). Significant upregulation of expression was detected for all of these genes in both roots and shoots, with expression was in all cases higher in the root tissues.
THE CASE OF SENESCENCE PROGRESSION, HYPOXIA AND PROLYL 4HYDROXYLASES
Despite the fact that modified and controlled atmosphere storage (hypoxia) has been used extensively by the food and horticulture industry for decades as a means of extending the storage life of fruits and vegetables, the molecular basis of this phenomenon is unknown. The cut carnation flower provides several advantages as a model system for study of hypoxia since exposure of carnation flowers to hypoxia does not present problems related to gas diffusion as occur in solid plant organs such as potato tubers and apples. Early stage response involves oxygen sensing and signal transduction of hypoxia. Initially, we identified hypoxia-induced carnation alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC) full-length cDNAs from hypoxic carnation petals and characterized their expression
[Owen, C.A, Spano,T., Hajjar, S. E, Tunaru, V., Harytunyan, S., Filali, L., and Kalaitzis, P (2004) Expression of genes for Alcohol Dehydrogenase and Pyruvate Decarboxylase in Petals of Cut Carnation Flowers in Response to Hypoxia and Anoxia (Physiologia Plantarum, 122:412-418].
To this direction we investigated the expression of carnation petal prolyl-4-hydroxylase homologue cDNA in response to hypoxic conditions. In hypoxia-treated Arabidopsis plants, members of the prolyl-4-hydroxylase homologues are up-regulated within the first hours of exposure. In mammals, prolyl-4-hydroxylases are considered members of the oxygen sensing machinery and are involved in the regulation of hypoxic response.
Real Time RT-PCR analysis was used to examine the expression of Dcp4h1 in the petals of carnation flowers exposed to a hypoxia time course. A transient 2-fold increase in the expression of Dcp4h1 was detected after 3 hours of exposure. After 6 hours of hypoxia the expression level of Dcp4h1 had returned to baseline levels.
AGROFOOD FORENSICS: THE CASE OF OLIVE OIL
Olive production is a significant land use in the Mediterranean countries with important environmental, social and economic considerations. Consumption of olive oil has been strongly connected to the Mediterranean diet which is widely considered to be healthy and has been adopted by people all over the world. Therefore brand name, high quality extra virgin olive oil is sold at high prices assuming it has passed stringent quality control. The varietal origin of olive oil is one of the most important factors associated to brand name premium quality. The development of cost-efficient, DNA-based protocols to identify varietal origin, composition and adulteration of olive oil will provide to the olive industry the means to protect their products against fraud and misdescription. This is the major objective of this work.
To this direction we initiated a project few years ago to fingerprint most of the commercial olive tree varieties of the Southeastern Mediterranean region from countries such as Greece, Turkey, Lebanon etc using the AFLP technology. High level of genetic variability was detected among the Greek cultivars. Similarly, we found that the Turkish germplasm had a relatively broad genetic background and, most importantly for breeding programs, it is distinct from the Greek germplasm [Owen et al., 2005. AFLP reveals structural details of genetic diversity within cultivated olive germplasm from the Easterm Mediterranean. Theoretical & Applied Genetics, in press).
We currently develop a PCR-based methodology to identify the varietal origin of olive oil based on known olive microsatellites (SSRs). We also develop a more advanced methodology based on SNPs (single nucleotide polymorphism) that will be identified in nuclear and chloroplastic genes.
Amplification of chloroplastic DNA from filtered olive oil samples taken from different brands of commercial olive oil sold in the local supermarkets. The varietal composition of olive oil will be determined using Real Time PCR while the efficiency of pyrosequencing will be addressed in the quantification and scaling-up aspects of the protocol.
DETECTION OF GMOs: THE CASE OF COTTON
The laboratory of Plant Biotechnology & GMO Testing provides laboratory services to the industry on qualitative and quantitative GMO testing in agrofood products, animal feed, fish food, olive oil and their products and any other product made of or consist of soya, maize, cotton etc.
DEVELOPMENT OF COTTON QUANTITATIVE STANDARDS
Cotton is a very important crop for Greece since is the largest cotton producer in the European Union. Therefore, there is a pressing need for GMO-Testing of cotton seeds and cotton products for the presence of GMOs. To this direction we initiated a project to develop a cost-efficient methodology for production of cotton quantitative standards for GMO analysis using the Real Time PCR methodology in collaboration with a private biotechnology company.
We have already identified a reference gene from the cotton genome that has been tested extensively in various other genomes and does not amplify PCR products indicating specificity for cotton. We have also developed mixtures of GM-cotton powder producing standards for 0.1%, 0.5%, 1%, 2% and 5% that are currently under evaluation.
The use of cotton reference gene-specific primers resulted on amplification of PCR product only from cotton genomic DNA and not from other plant species as shown in the figure.
Last update: 03 of February, 2012