Polyols are compounds that play various physiological roles in plants. Here we present the identi... more Polyols are compounds that play various physiological roles in plants. Here we present the identification of four cDNA clones of the model legume Lotus japonicus, encoding proteins of the monosaccharide transporter-like (MST) superfamily that share significant homology with previously characterized polyol transporters (PLTs). One of the transporters, named LjPLT4, was characterized functionally after expression in yeast. Transport assays revealed that LjPLT4 is a xylitol-specific H(+)-symporter (K (m), 0.34 mM). In contrast to the previously characterized homologues, LjPLT4 was unable to transport other polyols, including mannitol, sorbitol, myo-inositol and galactitol, or any of the monosaccharides tested. Interestingly, some monosaccharides, including fructose and xylose, inhibited xylitol uptake, although no significant uptake of these compounds was detected in the LjPLT4 transformed yeast cells, suggesting interactions with the xylitol binding site. Subcellular localization of LjPLT4-eYFP fusions expressed in Arabidopsis leaf epidermal cells indicated that LjPLT4 is localized in the plasma membrane. Real-time RT-PCR revealed that LjPLT4 is expressed in all major plant organs, with maximum transcript accumulation in leaves correlating with maximum xylitol levels there, as determined by GC-MS. Thus, LjPLT4 is the first plasma membrane xylitol-specific H(+)-symporter to be characterized in plants.
In the present work, we describe the characterisation of the glutathione transferase (GST) gene f... more In the present work, we describe the characterisation of the glutathione transferase (GST) gene family from Agrobacterium tumefaciens C58. A genome survey revealed the presence of eight GST-like proteins in A. tumefaciens (AtuGSTs). Comparison by multiple sequence alignment generated a dendrogram revealing the phylogenetic relationships of AtuGSTs-like proteins. The beta and theta classes identified in other bacterial species are represented by five members in A. tumefaciens C58. In addition, there are three "orphan" sequences that do not fit into any previously recognised GST classes. The eight GST-like genes were cloned, expressed in Escherichia coli and their substrate specificity was determined towards 17 different substrates. The results showed that AtuGSTs catalyse a broad range of reactions, with different members of the family exhibiting quite varied substrate specificity. The 3D structures of AtuGSTs were predicted using molecular modelling. The use of comparative sequence and structural analysis of the AtuGST isoenzymes allowed us to identify local sequence and structural characteristics between different GST isoenzymes and classes. Gene expression profiling was conducted under normal culture conditions as well as under abiotic stress conditions (addition of xenobiotics, osmotic stress and cold and heat shock) to induce and monitor early stress-response mechanisms. The results reveal the constitutive expression of GSTs in A. tumefaciens and a modulation of GST activity after treatments, indicating that AtuGSTs presumably participate in a wide range of functions, many of which are important in counteracting stress conditions. These functions may be relevant to maintaining cellular homeostasis as well as in the direct detoxification of toxic compounds.
To explore possible sources of transgenic resistance to the rhizomania-causing Beet necrotic yell... more To explore possible sources of transgenic resistance to the rhizomania-causing Beet necrotic yellow vein virus (BNYVV), Nicotiana benthamiana plants were constructed to express the harpin of Pseudomonas syringae pv. phaseolicola (HrpZ(Psph)). The HrpZ protein was expressed as an N-terminal fusion to the PR1 signal peptide (SP/HrpZ) to direct harpin accumulation to the plant apoplast. Transgene integration was verified by mPCR in all primary transformants (T0), while immunoblot analysis confirmed that the protein HrpZ(Psph) was produced and the signal peptide was properly processed. Neither T0 plants nor selfed progeny (T1) showed macroscopically visible necrosis or any other macroscopic phenotypes. However, plants expressing the SP/HrpZ(Psph) showed increased vigor and grew faster in comparison with non-transgenic control plants. Transgenic resistance was assessed after challenge inoculation with BNYVV on T1 progeny by scoring of disease symptoms and by DAS-ELISA at 20 and 30 dpi. T...
With the aim of achieving durable resistance against rhizomania disease of sugar beet, the employ... more With the aim of achieving durable resistance against rhizomania disease of sugar beet, the employment of different sources of resistance to Beet necrotic yellow vein virus was pursued. To this purpose, Nicotiana benthamiana transgenic plants that simultaneously produce dsRNA originating from a conserved region of the BNYVV replicase gene and the HrpZPsph protein in a secreted form (SP/HrpZPsph) were produced. The integration and expression of both transgenes as well as proper production of the harpin protein were verified in all primary transformants and selfed progeny (T1, T2). Transgenic resistance was assessed by BNYVV-challenge inoculation on T2 progeny by scoring disease symptoms and DAS-ELISA at 20 and 30 dpi. Transgenic lines possessing single transformation events for both transgenes as well as wild type plants were included in inoculation experiments. Transgenic plants were highly resistant to virus infection, whereas in some cases immunity was achieved. In all cases, the r...
Various types of sorghum were subjected to thermal stress to reveal the mode of expression of gen... more Various types of sorghum were subjected to thermal stress to reveal the mode of expression of genes of the heat shock protein ( hsp ) family. In silico sequence determination of hsp genes in related cereal species led to the selection of appropriate primers for PCR amplification of a segment corresponding to the hsp90 gene from sorghum. Deduced sequence information allowed the design of gene specific primers for quantification of hsp90 gene expression by means of real-time quantitative polymerase chain reaction (RTqPCR). Fourteen days-old plants were exposed to a temperature of 47°C for a time period ranging from 10 to 180 min. Total RNA was extracted from stressed and control plants and subjected to reverse transcription and RT-qPCR analysis. The actin gene was used as an internal standard. Gene expression was assessed by using cDNA from all types of plant material and for all the different durations of heat stress exposure. Data from RT-qPCR analyses were analyzed using REST softw...
Sweet sorghum's high yielding ability, low-input demands and tolerance to stresses, render it... more Sweet sorghum's high yielding ability, low-input demands and tolerance to stresses, render it highly suitable for syrup and bioenergy production. Exploiting its biomass, however is hampered by the seasonality of its production and the rapid post-harvest sugar catabolism degrading biomass quality. We aimed at elucidating aspects of sweet sorghum’s metabolism at different developmental and post-harvest stages, to investigate possibilities of expanding the harvesting window. GC–MS-mediated metabolic profiling was employed to monitor changes across growth stages and targeted transcriptomic analysis was used to determine the expression of genes involved in sucrose metabolism. Changes were studied both in the leaves and stems before, during and after the stage considered as optimum for harvest, whereas harvested stems were analyzed to determine post-harvest changes. Significant alterations in the levels of sugars, amino acids and organic acids were found, sugar levels attaining a maxi...
Sugar beet plants with typical rhizomania symptoms were collected from the five major cultivation... more Sugar beet plants with typical rhizomania symptoms were collected from the five major cultivation zones of Greece. The presence of Beet necrotic yellow vein virus (BNYVV), the primary causal agent of the disease, was ascertained by DAS-ELISA in 38 out of 40 fields surveyed and the positive samples were subsequently examined for the presence of other soil-borne viruses which are frequently associated with rhizomania, using a multiplex RT-PCR assay targeting BNYVV, Beet soilborne virus (BSBV) and Beet virus Q (BVQ). The occurrence of BSBV and BVQ was confirmed in 9 and 23 rhizomania- infected fields, respectively. In contrast to surveys conducted in other countries, the presence of BVQ prevailed throughout Greece in dual infections with BNYVV, whereas BSBV was restricted to rhizomaniainfected fields from only two sugarbeet cultivation areas. Nine of the samples tested were infected with all three viruses. BSBV was always found in triple infections. To our knowledge, this is the first ...
Drought has a negative impact on plant growth and is responsible for considerable crop yield lose... more Drought has a negative impact on plant growth and is responsible for considerable crop yield loses worldwide. Given the importance of improving yield under drought, the ability to select tolerant genetic material is a prerequisite in all relevant plant breeding activities. Lentil is an economically important crop which often suffers from inadequate soil moisture. In this study, seed germination potential and seedling growth were determined in various genotypes exposed to drought as a means to explore the possibility of identifying drought-tolerant germplasm at an early stage. Drought stress experiments were carried out using six lentil cultivars, representing local and imported germplasm. Stress was induced by varying concentrations of polyethylene glycol (PEG6000: 0%, 5%, 10% and 20%). Genotype performance was assessed on a daily basis and referred to germination percentage (%), seed water absorbance (%), seedling water content (%), shoot and root length (cm) and number of seedling...
Various types of sorghum were subjected to thermal stress to reveal the mode of expression of gen... more Various types of sorghum were subjected to thermal stress to reveal the mode of expression of genes of the heat shock protein (hsp) family. In silico sequence determination of hsp genes in related cereal species led to the selection of appropriate primers for PCR amplification of a segment corresponding to the hsp90 gene from sorghum. Deduced sequence information allowed the design of gene specific primers for quantification of hsp90 gene expression by means of real-time quantitative polymerase chain reaction (RT-qPCR). Fourteen days-old plants were exposed to a temperature of 47 C for a time period ranging from 10 to 180 min. Total RNA was extracted from stressed and control plants and subjected to reverse transcription and RT-qPCR analysis. The actin gene was used as an internal standard. Gene expression was assessed by using cDNA from all types of plant material and for all the different durations of heat stress exposure. Data from RT-qPCR analyses were analyzed using REST softwa...
Chickpea (Cicer arietinum L.) is one of the most important legume crops worldwide, with its impor... more Chickpea (Cicer arietinum L.) is one of the most important legume crops worldwide, with its importance mainly relying on its high protein content. Chickpea productivity is strictly threatened by abiotic stresses, of which drought exerts the most crucial role in terms of growth inhibition and yield losses encountered. Given that germination is a critical stage that is negatively affected by drought, the aim of this study was to estimate the genotypic variability among ten chickpea genotypes and to determine the seed germination and seedling growth ability under drought stress conditions. Seeds were subjected to water stress by polyethylene glycol (PEG-6000) at five stress levels (0, 5, 10, 20, 30 and 50% PEG). Germination percentage, seed water content, seed water absorbance, root and shoot development and seedling vigour index were evaluated. The analyses revealed significant genetic variability in relation to genotypic performance under drought stress. Drought significantly affecte...
Among abiotic stresses, drought is undoubtedly one of the most severe environmental factors for a... more Among abiotic stresses, drought is undoubtedly one of the most severe environmental factors for a wide range of major crops, leading to considerable yield and economic losses. The adverse effects in crop yield reflect the result of a series of morphological and physiological changes but also changes in signaling pathways, transcriptional and post-transcriptional regulation of stress-responsive genes, and metabolic adaptations. Despite the exhausting studies elucidating plants’ metabolic response to drought, there is a knowledge gap in the biochemical mechanisms governing drought tolerance in lentil (Lens culinaris Medik.). The present study aimed to determine the fluctuations of the metabolite profiles of lentil genotypes with contrasting drought tolerance to discover possible biomarkers for screening tolerant genotypes at early growth stages. Lentil seedlings were subjected to osmotic drought stress, induced by polyethylene glycol, at two stress levels (2.5% and 5.0% PEG-6000) for ...
Previous efforts to transform lentil have been considerably hampered by the crop’s recalcitrant n... more Previous efforts to transform lentil have been considerably hampered by the crop’s recalcitrant nature, giving rise to particularly low transformation and regeneration frequencies. This study aimed at optimizing an Agrobacterium rhizogenes-mediated transformation protocol for the generation of composite lentil plantlets, comprised of transgenic hairy roots and wild-type shoots. Transformation was performed by inoculating the cut hypocotyl of young lentil seedlings, while optimization involved the use of different bacterial strains, namely R1000, K599 and Arqua, and protocols differing in media composition with respect to the presence of acetosyringone and MES. Composite plantlets had a transgenic hairy root system characterized by an increased number of hairy roots at the hypocotyl proximal region, occasionally showing plagiotropic growth. Overall findings underline that transformation frequencies are subject to the bacterial strain, media composition as well as their combined effec...
Polyols are compounds that play various physiological roles in plants. Here we present the identi... more Polyols are compounds that play various physiological roles in plants. Here we present the identification of four cDNA clones of the model legume Lotus japonicus, encoding proteins of the monosaccharide transporter-like (MST) superfamily that share significant homology with previously characterized polyol transporters (PLTs). One of the transporters, named LjPLT4, was characterized functionally after expression in yeast. Transport assays revealed that LjPLT4 is a xylitol-specific H(+)-symporter (K (m), 0.34 mM). In contrast to the previously characterized homologues, LjPLT4 was unable to transport other polyols, including mannitol, sorbitol, myo-inositol and galactitol, or any of the monosaccharides tested. Interestingly, some monosaccharides, including fructose and xylose, inhibited xylitol uptake, although no significant uptake of these compounds was detected in the LjPLT4 transformed yeast cells, suggesting interactions with the xylitol binding site. Subcellular localization of LjPLT4-eYFP fusions expressed in Arabidopsis leaf epidermal cells indicated that LjPLT4 is localized in the plasma membrane. Real-time RT-PCR revealed that LjPLT4 is expressed in all major plant organs, with maximum transcript accumulation in leaves correlating with maximum xylitol levels there, as determined by GC-MS. Thus, LjPLT4 is the first plasma membrane xylitol-specific H(+)-symporter to be characterized in plants.
In the present work, we describe the characterisation of the glutathione transferase (GST) gene f... more In the present work, we describe the characterisation of the glutathione transferase (GST) gene family from Agrobacterium tumefaciens C58. A genome survey revealed the presence of eight GST-like proteins in A. tumefaciens (AtuGSTs). Comparison by multiple sequence alignment generated a dendrogram revealing the phylogenetic relationships of AtuGSTs-like proteins. The beta and theta classes identified in other bacterial species are represented by five members in A. tumefaciens C58. In addition, there are three "orphan" sequences that do not fit into any previously recognised GST classes. The eight GST-like genes were cloned, expressed in Escherichia coli and their substrate specificity was determined towards 17 different substrates. The results showed that AtuGSTs catalyse a broad range of reactions, with different members of the family exhibiting quite varied substrate specificity. The 3D structures of AtuGSTs were predicted using molecular modelling. The use of comparative sequence and structural analysis of the AtuGST isoenzymes allowed us to identify local sequence and structural characteristics between different GST isoenzymes and classes. Gene expression profiling was conducted under normal culture conditions as well as under abiotic stress conditions (addition of xenobiotics, osmotic stress and cold and heat shock) to induce and monitor early stress-response mechanisms. The results reveal the constitutive expression of GSTs in A. tumefaciens and a modulation of GST activity after treatments, indicating that AtuGSTs presumably participate in a wide range of functions, many of which are important in counteracting stress conditions. These functions may be relevant to maintaining cellular homeostasis as well as in the direct detoxification of toxic compounds.
To explore possible sources of transgenic resistance to the rhizomania-causing Beet necrotic yell... more To explore possible sources of transgenic resistance to the rhizomania-causing Beet necrotic yellow vein virus (BNYVV), Nicotiana benthamiana plants were constructed to express the harpin of Pseudomonas syringae pv. phaseolicola (HrpZ(Psph)). The HrpZ protein was expressed as an N-terminal fusion to the PR1 signal peptide (SP/HrpZ) to direct harpin accumulation to the plant apoplast. Transgene integration was verified by mPCR in all primary transformants (T0), while immunoblot analysis confirmed that the protein HrpZ(Psph) was produced and the signal peptide was properly processed. Neither T0 plants nor selfed progeny (T1) showed macroscopically visible necrosis or any other macroscopic phenotypes. However, plants expressing the SP/HrpZ(Psph) showed increased vigor and grew faster in comparison with non-transgenic control plants. Transgenic resistance was assessed after challenge inoculation with BNYVV on T1 progeny by scoring of disease symptoms and by DAS-ELISA at 20 and 30 dpi. T...
With the aim of achieving durable resistance against rhizomania disease of sugar beet, the employ... more With the aim of achieving durable resistance against rhizomania disease of sugar beet, the employment of different sources of resistance to Beet necrotic yellow vein virus was pursued. To this purpose, Nicotiana benthamiana transgenic plants that simultaneously produce dsRNA originating from a conserved region of the BNYVV replicase gene and the HrpZPsph protein in a secreted form (SP/HrpZPsph) were produced. The integration and expression of both transgenes as well as proper production of the harpin protein were verified in all primary transformants and selfed progeny (T1, T2). Transgenic resistance was assessed by BNYVV-challenge inoculation on T2 progeny by scoring disease symptoms and DAS-ELISA at 20 and 30 dpi. Transgenic lines possessing single transformation events for both transgenes as well as wild type plants were included in inoculation experiments. Transgenic plants were highly resistant to virus infection, whereas in some cases immunity was achieved. In all cases, the r...
Various types of sorghum were subjected to thermal stress to reveal the mode of expression of gen... more Various types of sorghum were subjected to thermal stress to reveal the mode of expression of genes of the heat shock protein ( hsp ) family. In silico sequence determination of hsp genes in related cereal species led to the selection of appropriate primers for PCR amplification of a segment corresponding to the hsp90 gene from sorghum. Deduced sequence information allowed the design of gene specific primers for quantification of hsp90 gene expression by means of real-time quantitative polymerase chain reaction (RTqPCR). Fourteen days-old plants were exposed to a temperature of 47°C for a time period ranging from 10 to 180 min. Total RNA was extracted from stressed and control plants and subjected to reverse transcription and RT-qPCR analysis. The actin gene was used as an internal standard. Gene expression was assessed by using cDNA from all types of plant material and for all the different durations of heat stress exposure. Data from RT-qPCR analyses were analyzed using REST softw...
Sweet sorghum's high yielding ability, low-input demands and tolerance to stresses, render it... more Sweet sorghum's high yielding ability, low-input demands and tolerance to stresses, render it highly suitable for syrup and bioenergy production. Exploiting its biomass, however is hampered by the seasonality of its production and the rapid post-harvest sugar catabolism degrading biomass quality. We aimed at elucidating aspects of sweet sorghum’s metabolism at different developmental and post-harvest stages, to investigate possibilities of expanding the harvesting window. GC–MS-mediated metabolic profiling was employed to monitor changes across growth stages and targeted transcriptomic analysis was used to determine the expression of genes involved in sucrose metabolism. Changes were studied both in the leaves and stems before, during and after the stage considered as optimum for harvest, whereas harvested stems were analyzed to determine post-harvest changes. Significant alterations in the levels of sugars, amino acids and organic acids were found, sugar levels attaining a maxi...
Sugar beet plants with typical rhizomania symptoms were collected from the five major cultivation... more Sugar beet plants with typical rhizomania symptoms were collected from the five major cultivation zones of Greece. The presence of Beet necrotic yellow vein virus (BNYVV), the primary causal agent of the disease, was ascertained by DAS-ELISA in 38 out of 40 fields surveyed and the positive samples were subsequently examined for the presence of other soil-borne viruses which are frequently associated with rhizomania, using a multiplex RT-PCR assay targeting BNYVV, Beet soilborne virus (BSBV) and Beet virus Q (BVQ). The occurrence of BSBV and BVQ was confirmed in 9 and 23 rhizomania- infected fields, respectively. In contrast to surveys conducted in other countries, the presence of BVQ prevailed throughout Greece in dual infections with BNYVV, whereas BSBV was restricted to rhizomaniainfected fields from only two sugarbeet cultivation areas. Nine of the samples tested were infected with all three viruses. BSBV was always found in triple infections. To our knowledge, this is the first ...
Drought has a negative impact on plant growth and is responsible for considerable crop yield lose... more Drought has a negative impact on plant growth and is responsible for considerable crop yield loses worldwide. Given the importance of improving yield under drought, the ability to select tolerant genetic material is a prerequisite in all relevant plant breeding activities. Lentil is an economically important crop which often suffers from inadequate soil moisture. In this study, seed germination potential and seedling growth were determined in various genotypes exposed to drought as a means to explore the possibility of identifying drought-tolerant germplasm at an early stage. Drought stress experiments were carried out using six lentil cultivars, representing local and imported germplasm. Stress was induced by varying concentrations of polyethylene glycol (PEG6000: 0%, 5%, 10% and 20%). Genotype performance was assessed on a daily basis and referred to germination percentage (%), seed water absorbance (%), seedling water content (%), shoot and root length (cm) and number of seedling...
Various types of sorghum were subjected to thermal stress to reveal the mode of expression of gen... more Various types of sorghum were subjected to thermal stress to reveal the mode of expression of genes of the heat shock protein (hsp) family. In silico sequence determination of hsp genes in related cereal species led to the selection of appropriate primers for PCR amplification of a segment corresponding to the hsp90 gene from sorghum. Deduced sequence information allowed the design of gene specific primers for quantification of hsp90 gene expression by means of real-time quantitative polymerase chain reaction (RT-qPCR). Fourteen days-old plants were exposed to a temperature of 47 C for a time period ranging from 10 to 180 min. Total RNA was extracted from stressed and control plants and subjected to reverse transcription and RT-qPCR analysis. The actin gene was used as an internal standard. Gene expression was assessed by using cDNA from all types of plant material and for all the different durations of heat stress exposure. Data from RT-qPCR analyses were analyzed using REST softwa...
Chickpea (Cicer arietinum L.) is one of the most important legume crops worldwide, with its impor... more Chickpea (Cicer arietinum L.) is one of the most important legume crops worldwide, with its importance mainly relying on its high protein content. Chickpea productivity is strictly threatened by abiotic stresses, of which drought exerts the most crucial role in terms of growth inhibition and yield losses encountered. Given that germination is a critical stage that is negatively affected by drought, the aim of this study was to estimate the genotypic variability among ten chickpea genotypes and to determine the seed germination and seedling growth ability under drought stress conditions. Seeds were subjected to water stress by polyethylene glycol (PEG-6000) at five stress levels (0, 5, 10, 20, 30 and 50% PEG). Germination percentage, seed water content, seed water absorbance, root and shoot development and seedling vigour index were evaluated. The analyses revealed significant genetic variability in relation to genotypic performance under drought stress. Drought significantly affecte...
Among abiotic stresses, drought is undoubtedly one of the most severe environmental factors for a... more Among abiotic stresses, drought is undoubtedly one of the most severe environmental factors for a wide range of major crops, leading to considerable yield and economic losses. The adverse effects in crop yield reflect the result of a series of morphological and physiological changes but also changes in signaling pathways, transcriptional and post-transcriptional regulation of stress-responsive genes, and metabolic adaptations. Despite the exhausting studies elucidating plants’ metabolic response to drought, there is a knowledge gap in the biochemical mechanisms governing drought tolerance in lentil (Lens culinaris Medik.). The present study aimed to determine the fluctuations of the metabolite profiles of lentil genotypes with contrasting drought tolerance to discover possible biomarkers for screening tolerant genotypes at early growth stages. Lentil seedlings were subjected to osmotic drought stress, induced by polyethylene glycol, at two stress levels (2.5% and 5.0% PEG-6000) for ...
Previous efforts to transform lentil have been considerably hampered by the crop’s recalcitrant n... more Previous efforts to transform lentil have been considerably hampered by the crop’s recalcitrant nature, giving rise to particularly low transformation and regeneration frequencies. This study aimed at optimizing an Agrobacterium rhizogenes-mediated transformation protocol for the generation of composite lentil plantlets, comprised of transgenic hairy roots and wild-type shoots. Transformation was performed by inoculating the cut hypocotyl of young lentil seedlings, while optimization involved the use of different bacterial strains, namely R1000, K599 and Arqua, and protocols differing in media composition with respect to the presence of acetosyringone and MES. Composite plantlets had a transgenic hairy root system characterized by an increased number of hairy roots at the hypocotyl proximal region, occasionally showing plagiotropic growth. Overall findings underline that transformation frequencies are subject to the bacterial strain, media composition as well as their combined effec...
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