Centre for Research and Technology Hellas - CERTH INSTITUTE OF AGROBIOTECHNOLOGY - INA 6th km. Charilaou-Thermi Road P.O. Box 60361, 57001 Thermi, Thessaloniki, Greece : + 30 2311 257531 : + 30 2310-498270 : http://www.certh.gr/ina.en.aspx
panmadi@hotmail.com
CURRENT POST 2011 Researcher (C) in The Centre for Research & Technology Hellas (CERTH), Institute of Agrobiotechnology (INA)
2008-2011 Researcher (D) in The Centre for Research & Technology Hellas (CERTH), Institute of Agrobiotechnology (INA)
EDUCATION 1999 – 2004 PhD “Development and analysis of transgenic plants” Aristotle University of Thessaloniki (A.U.TH.), Faculty of Agriculture. (Interrupted by 18 months compulsory Army Service) 1996-1998 Master of Science in “Genetic engineering and plant physiology” Aristotle University of Thessaloniki (A.U.TH.), Faculty of Agriculture. 1991-1996 Diploma in Agriculture. Grade 6,84 ( /10) One year undergraduate research project in the laboratory of genetics and plant breeding.“Evaluation of F4 families of hard wheat and performance of selected wheat lines.” Aristotle University of Thessaloniki (A.U.TH.), Faculty of Agriculture.
TEACHING 1 year teaching of Plant Breeding Lab in Technological Institute Of W. Macedonia, Florina
COMPETITIVE SCHOLARSHIPS AWARDED Marie Curie PhD Fellowship 11/11/2002 – 10/11/2003. IKY (State Scholarships Foundation) scholarship for Masters of Science and PhD 1997- 2002
CONFERENCE PRESENTATIONS 19-22/09/2011 First results on the overexpression of CSGSTU isoenzymes in transgenic tobacco plants Lo cicero L., Madesis P., Tsaftaris A., Lo piero A.R. Proceedings of the Joint Meeting AGI-SIBV-SIGA Assisi, Italy 29/9/10 “Comparative study of HCV core expression in plant nucleus and chloroplasts.” EPSO Meeting, Finland 19/10/2009 Algae: The Energy Supplier of the Future, CERTH, Thessaloniki, Greece 11/01/2007 “Reverse genetics analysis of recombination proteins in chloroplasts.” North West Plant Science Meeting. Lancaster University. 12/09/2006 “Reverse genetics analysis of plastid recombination proteins” Plastid Preview, University of Essex. 22/04/2006 EU-FP6 workshop. “Transgene integration and excision: analyses of transgenic plants with altered levels of gyrase and RecA protein.” Warsaw, Poland. 10/09/2005 EU-FP6 workshop. “Elucidating the plastid DNA recombination pathway using comparative genomics” Naples, Italy. 11/12/2004 EU-FP6 workshop. “Isolation of cDNAs encoding plastid recombination proteins”. Cambridge, UK. 2000 Workshop on “Genome Sequence and Comparative Analysis”. Thessaloniki, Greece. 1998 Presented poster. Annual Conference, The Greek Biological Society, Samos. 1998 Presented poster. Annual Conference, The Greek Plant Pathology Society. Athens.
SEMINARS 2010 7th Training Workshop “Computational genomics tools for exploring -omics data resources” 11th and Friday 12th March 2010, Thessaloniki, Greece 2006 «Mass Spectrometry Applications». University of Manchester. 2006 «Confocal Microscopy use and applications» Workshop University of Manchester. 2005 «Microarrays use and applications» Workshop University of Manchester. 1999 «Genome Sequence and Comparative Analysis». European Commission Directorate General for Science. Research and Development Biotechnology Programme, Thessaloniki Greece. 21 - 25 November. 1997 «Plant Biotechnology and Applications» (intensive course). Mediterenian Agronomic Institute of Chania, Crete, Greece May-June.
Member in Marie Curie Fellows Association (MCFA) Greek Plant Breeding and Genetics Societ Greek Society for Biology research Agriculture chamber
PROFESSIONAL EXPERTISE AND POSTIONS HELD I have hands-on knowledge of advanced molecular biology techniques, handling and manipulating DNA sequences, vector construction, cloning in E.coli, nuclear and plastid transformation of tobacco, pepper and sugarbeet using Agrobacterium tumefaciens or particle bombardment, expertise in plant tissue culture and plant breeding, RNA and protein manipulations and the analyses of genomes. My all round professional expertise has been developed during the tenure of a number of posts listed below:
01/10/2008 today Researcher D, The Center for Research and Thecnology Hellas /Institute of Agrobiotechnology .
Contract research projects 2011 Grant "THALIS" Glutathione transferases (GSTs): molecular tools for the development of basic and applied research in the fileds of green and red biotechnology. (INA Group Leader). 26/02/2007-30/09/2008 Postdoctoral Research Associate, Faculty of Life Sciences, The University of Manchester. BBSRC grant: “A genomic-based analysis of chloroplast replication/recombination/repair pathways using RNAi, proteomics and transplastomic plants (BB/E02445/1).” 26/02/2004-31/07/2007 Postdoctoral Research Associate, Faculty of Life Sciences, The University of Manchester. Supported by EU FP6. “Plastomics-a technology for improving human health” 2001-2004 Agricultural and financial projects. My role was to develop Agricultural and Financial knowledge to improve EU funding to local farmers 09/01/2002 – 31/06/02 Biology teacher in private school Aristoteleio University of Thessaloniki (A.U.TH.), Faculty of Agriculture, Department of Plant cultivation, Laboratory of genetics and plant breeding. 02/2000- 01/01“Molecular and biotechnological approaches for improving Cistus creticus spp. 05/1999-07/99 & 12/1999-01/2000 “HCV CORE gene expression in transgenic plants” 08/99-11/99 “Comparative sequence analyses of crop genes” MAICH- Mediterranean Agronomic Institute of Chania, Crete 18/07/94- 12/8/94. Placement in olive crop production and forestry department. Management of olive groves, landscape and pests.
Abstract: Legumes considered as one of the most important crops worldwide. Due to high price as a PDO product, 27
commercial products of ââFava Santorinisââ are often subjected to adulterations from other legume prod- 28
ucts coming from other Lathyrus or Vicia and Pisum species. Using plant DNA barcoding regions (trnL and 29
rpoC) coupled with High Resolution Melting (Bar-HRM) we have developed a method allowing us to 30
detect and authenticate PDO ââFava Santorinisââ. Bar-HRM proved to be a very sensitive tool able to geno- 31
type Lathyrus and its closed relative species and to detect admixtures, being sensitive enough to as low as 32
1:100 of non-ââFava Santorinisââ in ââFava Santorinisââ commercial products. In conclusion, Bar-HRM anal- 33
ysis can be a faster, with higher resolution and cost effectiveness alternative method to authenticate PDO 34
ââFava Santorinisââ and to quantitatively detect adulterations in ââFava Santorinisââ with other relative com- 35
mercial ââFavaââ food products.
Abstract: The Leguminoseae family includes several economically important genera like legumes. Legumes are the
second most important agricultural family after cereals in terms of harvested area and production.
Legumes are key elements in the Mediterranean diet and as winter annuals with nitrogen fixing capacity,
they are extremely important for sustainable agriculture worldwide.
We report here the application of the Bar-HRM (Barcode DNA High Resolution Melting) analysis
method with the universal plant DNA barcoding region trnL which allowed the identification, adulteration
and quantification of major Greek and Mediterranean in general bean species. Bar-HRM detected
Lupinus spp. adulterants in Glycine max flour as low as 1:100. Moreover, the method was coupled with
the rapid Phire PCR kit that does not require prior DNA purification. This makes the method a very fast
and effective tool for barcoding Legumes and particularly for the crops examined not only for their
authenticity but for quantitative detection of purity of their seeds or their processed food and feed
products.
Abstract: To further understand flowering and flower organs formation in the monocot crop saffron crocus (Crocus sativus L.), we cloned three APETALA2-like cDNA sequences of the AP2/ERF transcription factor family designated CsatAP2a/b/c as well as the respective promoter region sequences. Bioinformatics analysis with putative orthologous sequences from various plant species suggested that all three cDNA sequences encode for AP2-like proteins with the AP2 characteristic motifs and amino acids. Phylogenetically, the isolated sequences were closest to the AP2-like genes from Pisum sativum, Arabidopsis thaliana and Oryza sativa. Expression analysis indicated that the isolated C. sativus sequences were expressed in all the examined organs. Expression of CsatAP2a/b/c cDNAs was also compared in wild-type and mutant C. sativus flowers lacking stamens or carpels. Sequence analysis of the promoter revealed the presence of putative binding motifis for CCAAT, AP2 and LEAFY transcription factors indicative of regulation by developmental signals.
Abstract: Genes in the phosphatidyl-ethanolamine-binding protein (PEBP) family are instrumental in regulating the fate of meristems and flowering time. To investigate the role of these genes in the monocotyledonous plant Crocus (Crocus sativus L), an industrially important crop cultivated for its nutritional and medicinal properties, we have cloned and characterized a CENTRORADIALIS/TERMINAL FLOWER1 (CEN/TFL1) like gene, named CsatCEN/TFL1-like, the first reported CEN/TFL1 gene characterized from such a perennial geophyte. Sequence analysis revealed that CsatCEN/TFL1 shows high similarity to its homologous PEBP family genes CEN/TFL1, FT and MFT from a variety of plant species and maintains the same exon/intron organization. Phylogenetic analysis of the CsatCEN/TFL1 amino acid sequence confirmed that the isolated sequences belong to the CEN/TFL1 clade of the PEBP family. CsatCEN/TFL1 transcripts could be detected in corms, flower and flower organs but not in leaves. An alternative spliced transcript was also detected in the flower. Comparison of expression levels of CsatCEN/TFL1 and its alternative spliced transcript in wild type flower and a double flower mutant showed no significant differences. Overexpression of CsatCEN/TFL1 transcript in Arabidopsis tfl1 plants reversed the phenotype of early flowering and terminal flowering of the tfl1 plants to a normal one. Computational analysis of the obtained promoter sequences revealed, next to common binding motifs in CEN/TFL1-like genes as well as other flowering gene promoters, the presence of two CArG binding sites indicative of control of CEN/TFL1 by MADS-box transcription factors involved in crocus flowering and flower organ formation.
Abstract: Legume species are part of a very important agricultural family, second only to cereals. Their importance for sustainable agriculture worldwide comes from their nitrogen-fixing ability. They include mainly annual grain crops and also very important perennial forage and pasture species. Given their small size, seed admixture and adulteration are a common problem, lowering the forage value, creating weed components in the grassland and causing digestive problems to animals. Here we report the application of the Barcode-DNA High-Resolution Melting (Bar-HRM) analysis method using the universal nuclear plant DNA barcoding region ITS2 for the identification, adulteration and quantification of the main pasture species. Bar-HRM detected Medicago lupulina adulterants in Trifolium pratense seeds as low as 1:100. In conclusion, Bar-HRM analysis could be a faster with higher resolution and cost-effective alternative method to authenticate forage and pasture species and quantitatively detect the purity of their seeds or their feed products
Abstract: Legumes and common bean (Phaseolus vulgaris L.), in particular, are important crops worldwide, consumed either as dried seeds or fresh fruits. Correct identification of common bean varieties is important, in order to ensure food quality, safety and authenticity for consumers. Recently, DNA based methods, including molecular markers like microsatellites (SSR), have been developed for plant species or variety identification genotyping and for identification of their ingredients in the final food products. Here, we have applied High Resolution Melting (HRM) analysis coupled with four microsatellite markers to facilitate the identification of protected geographic indication (PGI) common bean variety 'Plake Megalosperma Prespon' ('PMP'). The four microsatellite loci used were informative and were used to generate a unique melting curve profile of microsatellites for each variety tested. These microsatellite markers enabled the distinction and identification of the PGI (common bean variety 'PMP'). Hence, this assay provided a flexible, cost-effective and closed-tube microsatellite genotyping method, well suited to varietal identification and authentication analysis in common beans.
Abstract: Cistus creticus L. ssp. creticus is a medicinal aromatic shrub native in Crete (Greece).
The protocol described in this paper provides optimal levels of growth regulators required to
obtain high regeneration rates of Cistus in vitro. Micropropagation has been achieved through
rapid proliferation of shoot-tips on Murashige and Skoog (MS) basal medium supplemented with
0.2 mg l-1 6-benzylaminopurine (BAP). After four weeks shoots were transferred to MS medium
without growth regulators for further development and rooting. The highest percentage of
regenerated shoots was obtained with 0.1 mg l-1 TDZ and 0.1 mg l-1 NAA after 4 weeks.
Elongation and rooting was readily achieved when multiple shoots more than 1 cm in length were
singled out and cultured on the MS medium without growth regulators. The plantlets were
successfully adapted and grew vigorously in greenhouse conditions. This is the first report of
shoot regeneration in the genus Cistus. The regeneration protocol developed in this study provides
a basis for further investigation of the medicinally active constituents of this elite medicinal plant.
Abstract: 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.
Abstract: Plant cytosolic glutathione transferases (GSTs) are an ancient enzyme superfamily with multiple and diverse
functions which are important in counteracting biotic and abiotic stress. GSTs play an important role in catalyzing the conjugation of xenobiotics and endogenous electrophilic compounds with glutathione (GSH), such as pesticides, chemical carcinogens, environmental pollutants, which leads to their detoxification. GSTs not only catalyze detoxification reactions but they are also involved in GSH-dependent isomerization reactions, in GSH-dependent reduction of organic hydroperoxides formed during oxidative stress, biosynthesis of sulfur-containing secondary metabolites, and exhibit thioltransferase and dehydroascorbate reductase activity. This review focuses on plant GSTs, and attempts to give an overview of the new insights into the catalytic function and structural biology of these enzymes.
Abstract: Plant glutathione transferases (GSTs) superfamily consists of multifunctional enzymes and forms a
major part of the plants herbicide detoxification enzyme network. The tau class GST isoenzyme from
soybean, GmGSTU4, exhibits catalytic activity towards the diphenyl ether herbicide fluorodifen and is
active as glutathione-dependent peroxidase. Transgenic tobacco plants from the cultivar Basmas were
generated via Agrobacterium transformation. The aim was to evaluate in planta, GmGSTU4 role in
detoxificating the diphenyl ether herbicides fluorodifen and oxyfluorfen and the chloroacetanilides
alachlor and metolachlor. Transgenic tobacco plants were verified by PCR and Southern blot
hybridization and expression of GmGSTU4 was determined by RT-PCR. Leaf extracts from transgenic
plants showed moderate increase in GST activity towards CDNB and a significant increase towards
fluorodifen. GmGSTU4 overexpressing plants when treated with 200μΠfluorodifen or oxyfluorfen
exhibited reduced relative electrolyte leakage compared to wild type plants. Moreover all GmGSTU4
overexpressing lines exhibited significant increased tolerance towards alachlor as it was expressed by
the greater shoot and root elongation, when grown in vitro at 7.5 mg/L alachlor compared to wild type
plants however their tolerance was reduced at 15 mg/L alachlor. No significant increased tolerance
was observed to metolachlor. These results confirm the contribution of the specific GmGSTU4
isoenzyme from soybean in the detoxification of fluorodifen and alachlor in tobacco providing the basis
towards the development of transgenic plants with improved phytoremediation capabilities for future
use in environmental cleanup of fluorodifen and alachlor.
Abstract: Hepatitis C virus (HCV) is a major disease agent affecting ~3% of the worldâs population. Expression in plant chloroplasts enables low-cost production of the conserved HCV core protein used in diagnostic tests to combat virus spread in developing countries with high infection rates. The bactericidal activity of the 21 kDa pre-core protein hinders cloning the core gene in plastid expression cassettes, which are active in bacteria due to the similarities between bacterial and plastid promoters and ribosome-binding-sites. This was overcome by using a topology-dependent expression cassette containing tandem rrn and psbA plastid promoters, whose activity was shown to be dependent on temperature. The viral core gene and a codon-optimised gene encoding a C-terminal truncated 16 kDa core polypeptide were expressed in tobacco chloroplasts. The codon-optimised gene increased monocistronic core mRNA levels by at least 2-fold and core polypeptides by over 5-fold, relative to the native viral gene. Expression of the 16 kDa core polypeptide was stable in leaves of different ages. Anti-core antibodies in HCV-infected human sera were detected by the 16 kDa core polypeptide in total leaf protein fractionated on western blots providing a first step towards developing a chloroplast-based HCV diagnostic method.
Abstract: The objective of this work was to study the stress tolerance and regeneration capability of transgenic pepper plants carrying a sod gene, encoding a tomato chloroplast-localized Cu/Zn SOD protein. The expression of the sod gene was confirmed by enzymatic staining following polyacrylamide gel electrophoresis (PAGE), revealing a ânovelâ band, which could represent a heterodimeric enzyme. Transgenic T1 and T2 progeny plants were exposed to different oxidative stresses including methyl viologen (MV) and drought and found to have an increased resistance to oxidative damage. Furthermore, the SOD carrying transgenic pepper plants showed increased levels of regeneration efficiency compared to the wild type pepper plants. Pepper is a recalcitrant species in terms of its in vitro regeneration ability but it could be extremely useful for the development of pharmaceuticals. This approach enables the extent use of pepper for genetic transformation and the production of high valuable products in plants particularly the large fruit varieties.
Abstract: Hepatitis C virus (HCV) is the major agent causing chronic liver disease. The core gene is the most conserved sequence in the HCV genome and proved immunoreactive when expressed in bacteria and antigenic in humans. In order to test the ability of plants to express the core gene for the production of core antigen, transgenic tobacco plants carrying the core gene were generated. The core protein was stably synthesized in T(0) and T(1) generations and was found to be immunoreactive, not only with anti-core polyclonal and monoclonal antibodies, but also was able to recognize the HCV virus in infected human serum. The prospects of producing a plant based vaccine and/or a food vaccine for this important virus are discussed.
Abstract: In eukaryotes, many genes were transferred to the nucleus from prokaryotic ancestors of the cytoplasmic organelles during endosymbiotic evolution. In plants, the transfer of genetic material from the plastid (chloroplast) and mitochondrion to the nucleus is a continuing process. The cellular location of a kanamycin resistance gene tailored for nuclear expression (35SneoSTLS2) was monitored in the progeny of reciprocal crosses of tobacco (Nicotiana tabacum) in which, at the start of the experiments, the reporter gene was confined either to the male or the female parental plastid genome. Among 146,000 progeny from crosses where the transplastomic parent was male, 13 transposition events were identified, whereas only one atypical transposition was identified in a screen of 273,000 transplastomic ovules. In a second experiment, a transplastomic beta-glucuronidase reporter gene, tailored to be expressed only in the nucleus, showed frequent stochastic expression that was confined to the cytoplasm in the somatic cells of several plant tissues. This gene was stably transferred in two out of 98,000 seedlings derived from a male transplastomic line crossed with a female wild type. These data demonstrate relocation of plastid DNA to the nucleus in both somatic and gametophytic tissue and reveal a large elevation of the frequency of transposition in the male germline. The results suggest a new explanation for the occurrence of uniparental inheritance in eukaryotes.
Abstract: Overexpression in Escherichia coli of a tau (U) class glutathione transferase (GST) from maize (Zea mays L.), termed ZmGSTU1, caused a reduction in heme levels and an accumulation of porphyrin precursors. This disruption was highly specific, with the expression of the closely related ZmGSTU2 or other maize GSTs having little effect. Expression in E. coli of a series of chimeric ZmGSTU1/ZmGSTU2 proteins identified domains responsible for disrupting porphyrin metabolism. In addition to known heme precursors, expression of ZmGSTU1 led to the accumulation of a novel glutathione conjugate of harderoporphyrin(ogen) (2,7,12,18-tetramethyl-3-vinylporphyrin-8,13,17-tripropionic acid). Using the related protoporphyrinogen as a substrate, conjugation could be shown to occur on one vinyl group and was actively catalyzed by the ZmGSTU. In plant transgenesis studies, the ZmGSTUs did not perturb porphyrin metabolism when expressed in the cytosol of Arabidopsis or tobacco. However, expression of a ZmGSTU1-ZmGSTU2 chimera in the chloroplasts of tobacco resulted in the accumulation of the harderoporphyrin(ogen)-glutathione conjugate observed in the expression studies in bacteria. Our results show that the well known ability of GSTs to act as ligand binding (ligandin) proteins of porphyrins in vitro results in highly specific interactions with porphyrinogen intermediates, which can be demonstrated in both plants and bacteria in vivo.
Abstract: Many phytopathogenic species of the fungus Cercospora produce cercosporin, a photoactivated perylenequinone toxin that belongs to a family of photosensitizers, which absorb light energy and produce extremely cytotoxic, reactive oxygen species. The cpd1 (cercosporin photosensitizer detoxification) gene of yeast (Saccharomyces cerevisiae), which encodes for a novel protein with significant similarity to the FAD-dependent pyridine nucleotide reductases, confers resistance to cercosporin when over-expressed in yeast. The aim of this work was to investigate the potential ability of cpd1 gene to confer resistance to cercosporin when expressed in tobacco plants (Nicotiana tabacum). Transgenic tobacco plants were produced using Agrobacterium tumefaciens, with cpd1 integrated as the gene of interest. We report here that expression of cpd1 gene in tobacco can mediate resistance to cercosporin. The involvement of cpd1 gene in the detoxification of the cercosporin reinforces previous observations, which suggested that resistance to cercosporin is mediated by a mechanism involving toxin reduction.
Abstract: Removal of marker genes improves the design of transgenic plants. Homologous recombination between direct repeats provides a simple method for excising marker genes after transgenic cells and shoots have been isolated. Efficient implementation of the method requires high rates of homologous recombination relative to illegitimate recombination pathways. The procedure works well in plastids where homologous recombination predominates. Marker genes are flanked by engineered direct repeats. The number and length of direct repeats flanking a marker gene influence excision rate. Excision is automatic and loss of the marker gene is controlled by selection alone. After transgenic cells have been isolated selection is removed allowing loss of the marker gene. Excision is a unidirectional process resulting in the rapid accumulation of high levels of marker-free plastid genomes. Cytoplasmic sorting of marker-free plastids from marker-containing plastids leads to the isolation of marker free plants. Marker-free plants can be isolated following vegetative propagation or among the progeny of sexual crosses.
