Fruit constitutes a major component of our diet, providing fiber, vitamins, minerals, and many ot... more Fruit constitutes a major component of our diet, providing fiber, vitamins, minerals, and many other phytonutrients that promote good health. Fleshy fruits, such as tomatoes, already contain high levels of several of these ingredients. Nevertheless, efforts have been invested in increasing and diversifying the content of phytonutrients, such as carotenoids and flavonoids, in tomato fruits. Increasing levels of phytonutrients, such as lycopene, is highly justified from the perspective of the lycopene extraction industry due to cost effectiveness reasons. Diversifying phytonutrients, in particular those that contribute to fruit color, could potentially provide an array of attractive colors to our diet. Our major goal was to devise a novel strategy for developing tomato fruits with enhanced levels of phytochemicals known to promote good health with special emphasis on lycopene content. A further important goal was to analyze global gene expression of selected genetic lines produced throughout this study in order is to dissect the molecular mechanisms regulating phytonutrients accumulation in the tomato fruit. To achieve these goals we proposed to: 1. combine, by classical breeding, engineered polyamine metabolism with photomorphogenic high pigment mutants in order generate tomato plant with exceptionally high levels of phytonutrients; 2. use gene transfer technology for genetic introduction of key genes that promote phytonutrient accumulation in the tomato fruit, 3. Analyze accumulation patterns of the phytonutrients in the tomato fruit during ripening; 4. Analyze global gene expression during fruit ripening in selected genotypes identified in objectives 1 and 2, and 5. Identify and analyze regulatory mechanisms of chloroplast disassembly and chromoplast formation. During the 3 years research period we have carried out most of the research activities laid out in the original proposal and our key conclusions are as follows: 1. the engineered polyamine metabolism strategy proposed by the US collaborators can not increase lycopene content either on its own or in combination with an hp mutant (hp-2ᵈᵍ); 2. The hp-2ᵈᵍ affects strongly the transcriptional profile of the tomato fruit showing a strong tendency for up- rather than down-regulation of genes, 3. Ontology assignment of these miss-regulated genes revealed a consistent up-regulation of genes related to chloroplast biogenesis and photosynthesis in hp-2ᵈᵍ mutants throughout fruit development; 4. A tendency for up-regulation was also usually observed in structural genes involved in phytonutrientbiosynthesis; however this up-regulation was not as consistent. 5. Microscopic observations revealed a significantly higher number of chloroplasts in pericarp cells of mature-green hp-2ᵈᵍ/hp-2ᵈᵍ fruits in comparison to their normal fully isogenic counterparts. 6. The relative abundance of chloroplasts could be observed from early stages of fruit development. Cumulatively these results suggest that: 1. the overproduction of secondary metabolites, characterizing hp-2ᵈᵍ/hp-2ᵈᵍ fruits, is more due to chloroplast number rather then to transcriptional activation of structural genes of the relevant metabolic pathways, and 2. The molecular trigger increasing metabolite levels in hp-2ᵈᵍ mutant fruits should be traced at early stage of fruit development.
General The final goal and overall objective of the current research has been to modify lipid hyd... more General The final goal and overall objective of the current research has been to modify lipid hydroperoxidation in order to create desirable phenotypes in two important crops, potato and tomato, which normally are exposed to abiotic stress associated with such oxidation. The specific original objectives were: (i) the roles of lipoxygenase (LOX) and phospholipids hydroperoxide glutathione peroxidase (PHGPx) in regulating endogenous levels of lipid peroxidation in plant tissues; (ii) the effect of modified lipid peroxidation on fruit ripening, tuber quality, crop productivity and abiotic stress tolerance; (iii) the effect of simultaneous reduction of LOX and increase of PHGPx activities on fruit ripening and tuber quality; and (iv) the role of lipid peroxidation on expression of specific genes. We proposed to accomplish the research goal by genetic engineering of the metabolic activities of LOX and PHGPx using regulatable and tissue specific promoters, and study of the relationships between these two consecutive enzymes in the metabolism and catabolism of phospholipids hydroperoxides. USA Significant progress was made in accomplishing all objectives of proposed research. Due to inability to regenerate tomato plants after transforming with 35S-PHGPx chimeric gene construct, the role of low catalase induced oxidative stress instead of PHGPx was evaluated on agronomical performance of tomato plant and fruit quality attributes. Effects of polyamine, that protects DNA from oxidative stress, were also evaluated. The transgenic plants under expressing lipoxygenase (LOX-sup) were crossed with catalase antisense (CAT-anti) plants or polyamine over producing plants (SAM-over) and the lines homozygous for the two transgenes were selected. Agronomical performance of these line showed that low catalase induced oxidative stress negatively affected growth and development of tomato plants and resulted in a massive change in fruit gene expression. These effects of low catalase activity induced oxidative stress, including the massive shift in gene expression, were greatly overcome by the low lipoxygenase activity. Collectively results show that oxidative stress plays significant role in plant growth including the fruit growth. These results also for the first time indicated that a crosstalk between oxidative stress and lipoxygenase regulated processes determine the outcome during plant growth and development. Israel Regarding PHGPx, most of the study has concentrated on the first and the last specific objectives, since it became evident that plant transformation with this gene is not obvious. Following inability to achieve efficient transformation of potato and tomato using a variety of promoters, model plant systems (tobacco and potato cell cultures, tobacco calli and plantlets, and Arabidopsis) were used to establish the factors and to study the obstacles which prohibited the regeneration of plants carrying the genetic machinery for overproduction of PHGPx. Our results clearly demonstrate that while genetic transformation and over-expression of PHGPx occurs in pre-developmental tissue stage (cell culture, calli clusters) or in completed plant (Arabidopsis), it is likely that over-expression of this enzyme before tissue differentiation is leading to a halt of the regeneration process. To support this assumption, experiments, in which genetic engineering of a point-mutated PHGPx gene enable transformation and over-expression in plants of PhSPY modified in its catalytic site and thus inactive enzymatically, were successfully carried out. These combined results strongly suggest, that if in fact, like in animals and as we established in vitro, the plant PHGPx exhibits PH peroxidase activity, these peroxides are vital for the organisms developmental process.
The plant pathogenic bacterium, Erwinia carotovora subsp. carotovora (Ecc), causes soft rot on a ... more The plant pathogenic bacterium, Erwinia carotovora subsp. carotovora (Ecc), causes soft rot on a number of plant species. The most striking characteristic of soft-rot Erwinias is the production of large amounts of extracellular pectolytic enzymes (2). Ecc produces several pectate lyases and polygalacturonases (7,8,10). In addition to pectic enzymes, several environmental components, especially anaerobic conditions, have been implicated in the development of severity of soft rot disease (3). During the last five years, several laboratories have cloned DNA sequences encoding pectolytic enzymes in order to clarify their role in the disease development (7,8,10, Handa et al. unpublished results). Isolated PL and PG can cause soft rot symptoms on potato tuber (7,8,10). However, an E. coli strain expressing both PL and PG from cloned genes caused only a limited tissue maceration of potato tuber (8), thus suggesting the involvement of other gene product(s) in the development of disease. At present, little is known about the nature of these gene products. To investigate the genetic factors responsible for the virulence of Ecc we have isolated and characterized a large number of nonpathogenic mutants by transposon mutagenesis. Partial characterization of some of these mutants is presented in this paper.
The current study was designed to achieve the following objectives: Studying the effect of diethy... more The current study was designed to achieve the following objectives: Studying the effect of diethyl nitrosamine (DEN) on rats’ liver; and the effect of jojoba oil on the liver enzymes and DNA content. Ad-ministration of DEN induced a significant increase in levels of serum ALT and AST. Administration of jojoba oil alone or with DEN caused significant decrease in serum levels of ALT and AST, compared with the control group through the experi-mental period. The level of lipid per oxi-dation (MDA) levels increased in DEN treated group. On the other hand animals treated with jojoba and DEN showed a decrease in lipid per oxidation. Compared with the DNA content showed that jojoba oil provoked a massive accumulation of cells in the three cell phases (G0/G1, S and G2/M), this led to the appearance of a diploid DNA content peak in the cell cycle experiments which is characterized to apoptotic cell population. The histological results revealed that jojoba oil improved the alterations caused b...
Fruit constitutes a major component of our diet, providing fiber, vitamins, minerals, and many ot... more Fruit constitutes a major component of our diet, providing fiber, vitamins, minerals, and many other phytonutrients that promote good health. Fleshy fruits, such as tomatoes, already contain high levels of several of these ingredients. Nevertheless, efforts have been invested in increasing and diversifying the content of phytonutrients, such as carotenoids and flavonoids, in tomato fruits. Increasing levels of phytonutrients, such as lycopene, is highly justified from the perspective of the lycopene extraction industry due to cost effectiveness reasons. Diversifying phytonutrients, in particular those that contribute to fruit color, could potentially provide an array of attractive colors to our diet. Our major goal was to devise a novel strategy for developing tomato fruits with enhanced levels of phytochemicals known to promote good health with special emphasis on lycopene content. A further important goal was to analyze global gene expression of selected genetic lines produced throughout this study in order is to dissect the molecular mechanisms regulating phytonutrients accumulation in the tomato fruit. To achieve these goals we proposed to: 1. combine, by classical breeding, engineered polyamine metabolism with photomorphogenic high pigment mutants in order generate tomato plant with exceptionally high levels of phytonutrients; 2. use gene transfer technology for genetic introduction of key genes that promote phytonutrient accumulation in the tomato fruit, 3. Analyze accumulation patterns of the phytonutrients in the tomato fruit during ripening; 4. Analyze global gene expression during fruit ripening in selected genotypes identified in objectives 1 and 2, and 5. Identify and analyze regulatory mechanisms of chloroplast disassembly and chromoplast formation. During the 3 years research period we have carried out most of the research activities laid out in the original proposal and our key conclusions are as follows: 1. the engineered polyamine metabolism strategy proposed by the US collaborators can not increase lycopene content either on its own or in combination with an hp mutant (hp-2ᵈᵍ); 2. The hp-2ᵈᵍ affects strongly the transcriptional profile of the tomato fruit showing a strong tendency for up- rather than down-regulation of genes, 3. Ontology assignment of these miss-regulated genes revealed a consistent up-regulation of genes related to chloroplast biogenesis and photosynthesis in hp-2ᵈᵍ mutants throughout fruit development; 4. A tendency for up-regulation was also usually observed in structural genes involved in phytonutrientbiosynthesis; however this up-regulation was not as consistent. 5. Microscopic observations revealed a significantly higher number of chloroplasts in pericarp cells of mature-green hp-2ᵈᵍ/hp-2ᵈᵍ fruits in comparison to their normal fully isogenic counterparts. 6. The relative abundance of chloroplasts could be observed from early stages of fruit development. Cumulatively these results suggest that: 1. the overproduction of secondary metabolites, characterizing hp-2ᵈᵍ/hp-2ᵈᵍ fruits, is more due to chloroplast number rather then to transcriptional activation of structural genes of the relevant metabolic pathways, and 2. The molecular trigger increasing metabolite levels in hp-2ᵈᵍ mutant fruits should be traced at early stage of fruit development.
General The final goal and overall objective of the current research has been to modify lipid hyd... more General The final goal and overall objective of the current research has been to modify lipid hydroperoxidation in order to create desirable phenotypes in two important crops, potato and tomato, which normally are exposed to abiotic stress associated with such oxidation. The specific original objectives were: (i) the roles of lipoxygenase (LOX) and phospholipids hydroperoxide glutathione peroxidase (PHGPx) in regulating endogenous levels of lipid peroxidation in plant tissues; (ii) the effect of modified lipid peroxidation on fruit ripening, tuber quality, crop productivity and abiotic stress tolerance; (iii) the effect of simultaneous reduction of LOX and increase of PHGPx activities on fruit ripening and tuber quality; and (iv) the role of lipid peroxidation on expression of specific genes. We proposed to accomplish the research goal by genetic engineering of the metabolic activities of LOX and PHGPx using regulatable and tissue specific promoters, and study of the relationships between these two consecutive enzymes in the metabolism and catabolism of phospholipids hydroperoxides. USA Significant progress was made in accomplishing all objectives of proposed research. Due to inability to regenerate tomato plants after transforming with 35S-PHGPx chimeric gene construct, the role of low catalase induced oxidative stress instead of PHGPx was evaluated on agronomical performance of tomato plant and fruit quality attributes. Effects of polyamine, that protects DNA from oxidative stress, were also evaluated. The transgenic plants under expressing lipoxygenase (LOX-sup) were crossed with catalase antisense (CAT-anti) plants or polyamine over producing plants (SAM-over) and the lines homozygous for the two transgenes were selected. Agronomical performance of these line showed that low catalase induced oxidative stress negatively affected growth and development of tomato plants and resulted in a massive change in fruit gene expression. These effects of low catalase activity induced oxidative stress, including the massive shift in gene expression, were greatly overcome by the low lipoxygenase activity. Collectively results show that oxidative stress plays significant role in plant growth including the fruit growth. These results also for the first time indicated that a crosstalk between oxidative stress and lipoxygenase regulated processes determine the outcome during plant growth and development. Israel Regarding PHGPx, most of the study has concentrated on the first and the last specific objectives, since it became evident that plant transformation with this gene is not obvious. Following inability to achieve efficient transformation of potato and tomato using a variety of promoters, model plant systems (tobacco and potato cell cultures, tobacco calli and plantlets, and Arabidopsis) were used to establish the factors and to study the obstacles which prohibited the regeneration of plants carrying the genetic machinery for overproduction of PHGPx. Our results clearly demonstrate that while genetic transformation and over-expression of PHGPx occurs in pre-developmental tissue stage (cell culture, calli clusters) or in completed plant (Arabidopsis), it is likely that over-expression of this enzyme before tissue differentiation is leading to a halt of the regeneration process. To support this assumption, experiments, in which genetic engineering of a point-mutated PHGPx gene enable transformation and over-expression in plants of PhSPY modified in its catalytic site and thus inactive enzymatically, were successfully carried out. These combined results strongly suggest, that if in fact, like in animals and as we established in vitro, the plant PHGPx exhibits PH peroxidase activity, these peroxides are vital for the organisms developmental process.
The plant pathogenic bacterium, Erwinia carotovora subsp. carotovora (Ecc), causes soft rot on a ... more The plant pathogenic bacterium, Erwinia carotovora subsp. carotovora (Ecc), causes soft rot on a number of plant species. The most striking characteristic of soft-rot Erwinias is the production of large amounts of extracellular pectolytic enzymes (2). Ecc produces several pectate lyases and polygalacturonases (7,8,10). In addition to pectic enzymes, several environmental components, especially anaerobic conditions, have been implicated in the development of severity of soft rot disease (3). During the last five years, several laboratories have cloned DNA sequences encoding pectolytic enzymes in order to clarify their role in the disease development (7,8,10, Handa et al. unpublished results). Isolated PL and PG can cause soft rot symptoms on potato tuber (7,8,10). However, an E. coli strain expressing both PL and PG from cloned genes caused only a limited tissue maceration of potato tuber (8), thus suggesting the involvement of other gene product(s) in the development of disease. At present, little is known about the nature of these gene products. To investigate the genetic factors responsible for the virulence of Ecc we have isolated and characterized a large number of nonpathogenic mutants by transposon mutagenesis. Partial characterization of some of these mutants is presented in this paper.
The current study was designed to achieve the following objectives: Studying the effect of diethy... more The current study was designed to achieve the following objectives: Studying the effect of diethyl nitrosamine (DEN) on rats’ liver; and the effect of jojoba oil on the liver enzymes and DNA content. Ad-ministration of DEN induced a significant increase in levels of serum ALT and AST. Administration of jojoba oil alone or with DEN caused significant decrease in serum levels of ALT and AST, compared with the control group through the experi-mental period. The level of lipid per oxi-dation (MDA) levels increased in DEN treated group. On the other hand animals treated with jojoba and DEN showed a decrease in lipid per oxidation. Compared with the DNA content showed that jojoba oil provoked a massive accumulation of cells in the three cell phases (G0/G1, S and G2/M), this led to the appearance of a diploid DNA content peak in the cell cycle experiments which is characterized to apoptotic cell population. The histological results revealed that jojoba oil improved the alterations caused b...
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