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David Granot - One of the best experts on this subject based on the ideXlab platform.
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Constitutively overexpressing a tomato Fructokinase gene (LeFRK1) in cotton (Gossypium hirsutum L. cv. Coker 312) positively affects plant vegetative growth, boll number and seed cotton yield.
Functional plant biology : FPB, 2015Co-Authors: Thiya Mukherjee, David Granot, Yoshinori Kanayama, Mariana Ivanova, Marisela Dagda, A. Scott HoladayAbstract:Increasing Fructokinase (FRK) activity in cotton (Gossypium hirsutum L.) plants may reduce fructose inhibition of sucrose synthase (Sus) and lead to improved fibre yield and quality. Cotton was transformed with a tomato (Solanum lycopersicum L.) Fructokinase gene (LeFRK1) under the control of the CMV 35S promoter. In a greenhouse, the LeFRK1 plants had increased fibre and leaf FRK activity over nonexpressing nulls, but not improved fibre length and strength. Compared with the nulls, LeFRK1 plants yielded 13–100% more seed-cotton mass per boll and more bolls per plant, and therefore more seed cotton and fibre yield per plant. The enhanced yield was related to a greater seed number per boll for LeFRK1 plants. Photosynthetic rates were not appreciably different among genotypes. However, more area per leaf and leaf number (in some instances) for LeFRK1 plants than for nulls enhanced the capacity for C gain. Larger leaf areas for LeFRK1 plants were associated with larger stem diameters. Lower sucrose levels in developing leaves of LeFRK1 plants suggest that LeFRK1 overexpression leads to improved in vivo Sus activity in developing leaves and possibly in developing seeds. The improvement in yield for LeFRK1 plants may also be the result of improvements in photosynthate supply as a consequence of greater leaf area.
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Evidence for intracellular spatial separation of hexokinases and Fructokinases in tomato plants.
Planta, 2006Co-Authors: Hila Damari-weissler, Michal Kandel-kfir, David Gidoni, Anahit Mett, Eddy Belausov, David GranotAbstract:Four hexokinase (LeHXK1–4) and four Fructokinase (LeFRK1–4) genes were identified in tomato plants. Previous GFP fusion studies indicate that the gene product of LeHXK3 is associated with the mitochondria while that of LeHXK4 is located within plastids. In this study we found that the enzyme encoded by the Fructokinase gene LeFRK3 is also located within plastids. The presence of LeFrk3 enzyme in plastids raises the question of the origin of fructose in these organelles. The other three FRKs enzymes, LeFrk1&2&4, are located in the cytosol. Unlike LeFrk1&2&4, the two additional HXKs, LeHxk1&2, share a common membrane anchor domain and are associated with the mitochondria similar to LeHxk3. The difference in the locations of the cytoplasmic FRK and HXK isozymes suggests that glucose phosphorylation is confined to defined special intracellular localizations while fructose phosphorylation is less confined.
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Cloning, expression and characterization of LeFRK3, the fourth tomato (Lycopersicon esculentum Mill.) gene encoding Fructokinase☆
Plant Science, 2004Co-Authors: Marcelo A. German, Arthur A. Schaffer, Marina Petreikov, Israel Asher, Nir Dai, David GranotAbstract:Abstract A full-length cDNA encoding a novel fourth Fructokinase, LeFRK3, was cloned from green tomato (Lycopersicon esculentum Mill.) fruits. The putative protein shares 70, 65.5 and 69% amino acid homology with the three previously identified tomato Fructokinases encoded by LeFRK1, LeFRK2 and LeFRK4, respectively. This fourth Fructokinase has signature patterns of the pfkB family of carbohydrate kinases as well as substrate recognition sites and an ATP-binding domain. Confirmation for its Fructokinase activity was obtained by complementation of triple mutant yeast cells that are unable to phosphorylate or grow on either glucose or fructose as LeFRK3 complemented growth on fructose but not on glucose. Moreover, soluble crude protein extracts prepared from the transformed yeast cells revealed fructose but not glucose phosphorylation activity. In contrast to the LeFRK1 gene product which is inhibited neither by fructose nor by Mg, and to LeFRK2 gene product which is inhibited by both fructose and Mg, the LeFRK3 product is inhibited by fructose but not by Mg. Separation by HPLC-ion exchange chromatography pointed to the gene product of LeFRK3 as the protein responsible for the third peak of Fructokinase activity (FKIII), sharing the same pattern of fructose inhibition previously identified with FKIII in tomato fruits. Mapping of tomato Fructokinases indicated that LeFRK3 is located on chromosome 2, unlike LeFRK1 (chromosome 3), LeFRK2 (chromosome 6), and LeFRK4 (chromosome 10). The relative expression levels of the four known FRK genes in different tomato organs were analyzed by quantitative RT-PCR. LeFRK2 and LeFRK3 are the predominant genes expressed in all organs with LeFRK3 having the highest level of expression in leaves and apices. LeFRK4 is expressed only in stamens. This differential expression patterns combined with the different biochemical characteristics of the four FRK isozymes suggest that each plays a different role in plant development.
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suppression of Fructokinase encoded by lefrk2 in tomato stem inhibits growth and causes wilting of young leaves
Plant Journal, 2003Co-Authors: Marcelo A. German, Arthur A. Schaffer, Marina Petreikov, Nir Dai, Tanya Matsevitz, Rana Hanael, Nirit Bernstein, Marina Ioffe, Yosepha Shahak, David GranotAbstract:Summary Fructokinases catalyze the key step of fructose phosphorylation in plants. LeFRK2, the major Fructokinase-encoding gene in tomato plants, is abundantly expressed in roots, stems, and fruits. To analyze the role of LeFRK2 in plant development, we analyzed transgenic tomato plants with sense and antisense expression of StFRK, the potato homolog of LeFRK2. Increased Fructokinase activity had no effect. However, plants in which LeFRK2 was specifically suppressed, either via antisense suppression or via co-suppression, exhibited growth inhibition and wilting of young leaves at daytime. Grafting experiments indicated that a stem interstock of antisense plants was sufficient to inhibit growth and cause leaf wilting. Stem secondary xylem exhibited particular suppression of LeFRK2 and the area of active xylem, estimated by eosin uptake, was significantly smaller in antisense stem compared to that of wild-type plants. These results suggest that LeFRK2 might be required for proper development of xylem that affected growth and wilting.
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LeFRK4, a novel tomato (Lycopersicon esculentum Mill.) Fructokinase specifically expressed in stamens
Plant Science, 2002Co-Authors: Marcelo A. German, Arthur A. Schaffer, Nir Dai, Inna Chmelnitsky, Irina Sobolev, Yehiam Salts, Rivka Barg, David GranotAbstract:Abstract A full-length cDNA clone encoding a novel Fructokinase, LeFRK4, was isolated from a tomato (Lycopersicon esculentum Mill.) flower cDNA library. The putative protein shares 62.6 and 55.5% amino acid identity with the two known tomato Fructokinases, LeFRK1 and LeFRK2, respectively, and possesses three signature patterns of the pfkB family of carbohydrate kinases, two substrate recognition sites and an ATP-binding domain. The identification of LeFRK4 as a Fructokinase was confirmed by complementation of mutant yeast cells unable to phosphorylate or grow on either glucose or fructose. LeFRK4 complemented growth on fructose but not on glucose. Non-soluble crude protein extracts prepared from the transformed yeast cells exhibited fructose but not glucose phosphorylation activity. Expression analysis demonstrated that LeFRK4 is specifically expressed in stamens. Using quantitative RT-PCR, we examined the relative expression of the three known FRK genes in different tomato organs. LeFRK2, encoding a substrate-inhibited Fructokinase, was by far the predominantly expressed FRK gene in all organs, except for flowers in which it shared the same expression level with LeFRK4. The exclusive expression of LeFRK4 in stamens may point to a specific role of LeFRK4 in pollen development, anthesis, and perhaps pollination.
Arthur A. Schaffer - One of the best experts on this subject based on the ideXlab platform.
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Cloning, expression and characterization of LeFRK3, the fourth tomato (Lycopersicon esculentum Mill.) gene encoding Fructokinase☆
Plant Science, 2004Co-Authors: Marcelo A. German, Arthur A. Schaffer, Marina Petreikov, Israel Asher, Nir Dai, David GranotAbstract:Abstract A full-length cDNA encoding a novel fourth Fructokinase, LeFRK3, was cloned from green tomato (Lycopersicon esculentum Mill.) fruits. The putative protein shares 70, 65.5 and 69% amino acid homology with the three previously identified tomato Fructokinases encoded by LeFRK1, LeFRK2 and LeFRK4, respectively. This fourth Fructokinase has signature patterns of the pfkB family of carbohydrate kinases as well as substrate recognition sites and an ATP-binding domain. Confirmation for its Fructokinase activity was obtained by complementation of triple mutant yeast cells that are unable to phosphorylate or grow on either glucose or fructose as LeFRK3 complemented growth on fructose but not on glucose. Moreover, soluble crude protein extracts prepared from the transformed yeast cells revealed fructose but not glucose phosphorylation activity. In contrast to the LeFRK1 gene product which is inhibited neither by fructose nor by Mg, and to LeFRK2 gene product which is inhibited by both fructose and Mg, the LeFRK3 product is inhibited by fructose but not by Mg. Separation by HPLC-ion exchange chromatography pointed to the gene product of LeFRK3 as the protein responsible for the third peak of Fructokinase activity (FKIII), sharing the same pattern of fructose inhibition previously identified with FKIII in tomato fruits. Mapping of tomato Fructokinases indicated that LeFRK3 is located on chromosome 2, unlike LeFRK1 (chromosome 3), LeFRK2 (chromosome 6), and LeFRK4 (chromosome 10). The relative expression levels of the four known FRK genes in different tomato organs were analyzed by quantitative RT-PCR. LeFRK2 and LeFRK3 are the predominant genes expressed in all organs with LeFRK3 having the highest level of expression in leaves and apices. LeFRK4 is expressed only in stamens. This differential expression patterns combined with the different biochemical characteristics of the four FRK isozymes suggest that each plays a different role in plant development.
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suppression of Fructokinase encoded by lefrk2 in tomato stem inhibits growth and causes wilting of young leaves
Plant Journal, 2003Co-Authors: Marcelo A. German, Arthur A. Schaffer, Marina Petreikov, Nir Dai, Tanya Matsevitz, Rana Hanael, Nirit Bernstein, Marina Ioffe, Yosepha Shahak, David GranotAbstract:Summary Fructokinases catalyze the key step of fructose phosphorylation in plants. LeFRK2, the major Fructokinase-encoding gene in tomato plants, is abundantly expressed in roots, stems, and fruits. To analyze the role of LeFRK2 in plant development, we analyzed transgenic tomato plants with sense and antisense expression of StFRK, the potato homolog of LeFRK2. Increased Fructokinase activity had no effect. However, plants in which LeFRK2 was specifically suppressed, either via antisense suppression or via co-suppression, exhibited growth inhibition and wilting of young leaves at daytime. Grafting experiments indicated that a stem interstock of antisense plants was sufficient to inhibit growth and cause leaf wilting. Stem secondary xylem exhibited particular suppression of LeFRK2 and the area of active xylem, estimated by eosin uptake, was significantly smaller in antisense stem compared to that of wild-type plants. These results suggest that LeFRK2 might be required for proper development of xylem that affected growth and wilting.
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LeFRK4, a novel tomato (Lycopersicon esculentum Mill.) Fructokinase specifically expressed in stamens
Plant Science, 2002Co-Authors: Marcelo A. German, Arthur A. Schaffer, Nir Dai, Inna Chmelnitsky, Irina Sobolev, Yehiam Salts, Rivka Barg, David GranotAbstract:Abstract A full-length cDNA clone encoding a novel Fructokinase, LeFRK4, was isolated from a tomato (Lycopersicon esculentum Mill.) flower cDNA library. The putative protein shares 62.6 and 55.5% amino acid identity with the two known tomato Fructokinases, LeFRK1 and LeFRK2, respectively, and possesses three signature patterns of the pfkB family of carbohydrate kinases, two substrate recognition sites and an ATP-binding domain. The identification of LeFRK4 as a Fructokinase was confirmed by complementation of mutant yeast cells unable to phosphorylate or grow on either glucose or fructose. LeFRK4 complemented growth on fructose but not on glucose. Non-soluble crude protein extracts prepared from the transformed yeast cells exhibited fructose but not glucose phosphorylation activity. Expression analysis demonstrated that LeFRK4 is specifically expressed in stamens. Using quantitative RT-PCR, we examined the relative expression of the three known FRK genes in different tomato organs. LeFRK2, encoding a substrate-inhibited Fructokinase, was by far the predominantly expressed FRK gene in all organs, except for flowers in which it shared the same expression level with LeFRK4. The exclusive expression of LeFRK4 in stamens may point to a specific role of LeFRK4 in pollen development, anthesis, and perhaps pollination.
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LeFRK2, the gene encoding the major Fructokinase in tomato fruits, is not required for starch biosynthesis in developing fruits
Plant Science, 2002Co-Authors: Nir Dai, Arthur A. Schaffer, Marina Petreikov, Marcelo A. German, Tanya Matsevitz, Rana Hanael, Dvora Swartzberg, Yelena Yeselson, David GranotAbstract:Abstract The LeFRK2 gene product has been proposed to play a role in transient starch accumulation in developing tomato (Lycopersicon esculentum) fruits. To analyze the role of LeFRK2 in starch biosynthesis, we produced transgenic tomato plants with sense and antisense expression of StFRK, the potato homologue to LeFRK2. Fruits of homozygous plants expressing sense or antisense StFRK exhibited suppression of LeFRK2, concomitantly with specific elimination of the FKI isozyme, indicating that FKI is the gene product of LeFRK2. The activity of Fructokinase was reduced in antisense and sense homozygous fruits and increased in fruits of sense hemizygous plants. The modified activities of Fructokinase led to small but significant changes both in the steady state levels of sugars and in the level of phosphorylated sugars in fruits. However, fruits lacking FKI had increased rather than decreased starch content. We therefore concluded that LeFRK2 is not required for transient starch accumulation in tomato fruits. Rather, LeFRK2 might have a role in the regulation of sucrose import into tomato fruits.
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Characterization of native and yeast-expressed tomato fruit Fructokinase enzymes
Phytochemistry, 2001Co-Authors: Marina Petreikov, Nir Dai, David Granot, Arthur A. SchafferAbstract:Three Fructokinase isozymes (FKI, FKII, FKIII) were separated from both immature and ripe tomato fruit pericarp. All three isozymes were specific for fructose with undetectable activity towards glucose or mannose. The three isozymes could be distinguished from one another with respect to response to fructose, Mg and nucleotide donor concentrations and this allowed the comparison of the fruit enzymes with the gene products of the two known cloned tomato Fructokinase genes, LeFRK1 and LeFRK2. FKI was characterized by both substrate (fructose), as well as Mg, inhibition; FKII was inhibited by neither fructose nor Mg; and FKIII was inhibited by fructose but not by Mg. ATP was the preferred nucleotide donor for all three FKs and FKI showed inhibition by CTP and GTP above 1 mM. All three FKs showed competitive inhibition by ADP. During the maturation of the tomato fruit total FK activity decreased dramatically. There were decreases in activity of all three FKs, nevertheless, all were still observed in the ripe fruit. The two tomato LeFRK genes were expressed in yeast and the gene products were characterized with respect to the distinguishing characteristics of fructose, Mg and nucleotide inhibition. Our results indicate that FKI is the gene product of LeFRK2 and FKII is probably the gene product of LeFRK1.
Marina Petreikov - One of the best experts on this subject based on the ideXlab platform.
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Cloning, expression and characterization of LeFRK3, the fourth tomato (Lycopersicon esculentum Mill.) gene encoding Fructokinase☆
Plant Science, 2004Co-Authors: Marcelo A. German, Arthur A. Schaffer, Marina Petreikov, Israel Asher, Nir Dai, David GranotAbstract:Abstract A full-length cDNA encoding a novel fourth Fructokinase, LeFRK3, was cloned from green tomato (Lycopersicon esculentum Mill.) fruits. The putative protein shares 70, 65.5 and 69% amino acid homology with the three previously identified tomato Fructokinases encoded by LeFRK1, LeFRK2 and LeFRK4, respectively. This fourth Fructokinase has signature patterns of the pfkB family of carbohydrate kinases as well as substrate recognition sites and an ATP-binding domain. Confirmation for its Fructokinase activity was obtained by complementation of triple mutant yeast cells that are unable to phosphorylate or grow on either glucose or fructose as LeFRK3 complemented growth on fructose but not on glucose. Moreover, soluble crude protein extracts prepared from the transformed yeast cells revealed fructose but not glucose phosphorylation activity. In contrast to the LeFRK1 gene product which is inhibited neither by fructose nor by Mg, and to LeFRK2 gene product which is inhibited by both fructose and Mg, the LeFRK3 product is inhibited by fructose but not by Mg. Separation by HPLC-ion exchange chromatography pointed to the gene product of LeFRK3 as the protein responsible for the third peak of Fructokinase activity (FKIII), sharing the same pattern of fructose inhibition previously identified with FKIII in tomato fruits. Mapping of tomato Fructokinases indicated that LeFRK3 is located on chromosome 2, unlike LeFRK1 (chromosome 3), LeFRK2 (chromosome 6), and LeFRK4 (chromosome 10). The relative expression levels of the four known FRK genes in different tomato organs were analyzed by quantitative RT-PCR. LeFRK2 and LeFRK3 are the predominant genes expressed in all organs with LeFRK3 having the highest level of expression in leaves and apices. LeFRK4 is expressed only in stamens. This differential expression patterns combined with the different biochemical characteristics of the four FRK isozymes suggest that each plays a different role in plant development.
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suppression of Fructokinase encoded by lefrk2 in tomato stem inhibits growth and causes wilting of young leaves
Plant Journal, 2003Co-Authors: Marcelo A. German, Arthur A. Schaffer, Marina Petreikov, Nir Dai, Tanya Matsevitz, Rana Hanael, Nirit Bernstein, Marina Ioffe, Yosepha Shahak, David GranotAbstract:Summary Fructokinases catalyze the key step of fructose phosphorylation in plants. LeFRK2, the major Fructokinase-encoding gene in tomato plants, is abundantly expressed in roots, stems, and fruits. To analyze the role of LeFRK2 in plant development, we analyzed transgenic tomato plants with sense and antisense expression of StFRK, the potato homolog of LeFRK2. Increased Fructokinase activity had no effect. However, plants in which LeFRK2 was specifically suppressed, either via antisense suppression or via co-suppression, exhibited growth inhibition and wilting of young leaves at daytime. Grafting experiments indicated that a stem interstock of antisense plants was sufficient to inhibit growth and cause leaf wilting. Stem secondary xylem exhibited particular suppression of LeFRK2 and the area of active xylem, estimated by eosin uptake, was significantly smaller in antisense stem compared to that of wild-type plants. These results suggest that LeFRK2 might be required for proper development of xylem that affected growth and wilting.
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LeFRK2, the gene encoding the major Fructokinase in tomato fruits, is not required for starch biosynthesis in developing fruits
Plant Science, 2002Co-Authors: Nir Dai, Arthur A. Schaffer, Marina Petreikov, Marcelo A. German, Tanya Matsevitz, Rana Hanael, Dvora Swartzberg, Yelena Yeselson, David GranotAbstract:Abstract The LeFRK2 gene product has been proposed to play a role in transient starch accumulation in developing tomato (Lycopersicon esculentum) fruits. To analyze the role of LeFRK2 in starch biosynthesis, we produced transgenic tomato plants with sense and antisense expression of StFRK, the potato homologue to LeFRK2. Fruits of homozygous plants expressing sense or antisense StFRK exhibited suppression of LeFRK2, concomitantly with specific elimination of the FKI isozyme, indicating that FKI is the gene product of LeFRK2. The activity of Fructokinase was reduced in antisense and sense homozygous fruits and increased in fruits of sense hemizygous plants. The modified activities of Fructokinase led to small but significant changes both in the steady state levels of sugars and in the level of phosphorylated sugars in fruits. However, fruits lacking FKI had increased rather than decreased starch content. We therefore concluded that LeFRK2 is not required for transient starch accumulation in tomato fruits. Rather, LeFRK2 might have a role in the regulation of sucrose import into tomato fruits.
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Characterization of native and yeast-expressed tomato fruit Fructokinase enzymes
Phytochemistry, 2001Co-Authors: Marina Petreikov, Nir Dai, David Granot, Arthur A. SchafferAbstract:Three Fructokinase isozymes (FKI, FKII, FKIII) were separated from both immature and ripe tomato fruit pericarp. All three isozymes were specific for fructose with undetectable activity towards glucose or mannose. The three isozymes could be distinguished from one another with respect to response to fructose, Mg and nucleotide donor concentrations and this allowed the comparison of the fruit enzymes with the gene products of the two known cloned tomato Fructokinase genes, LeFRK1 and LeFRK2. FKI was characterized by both substrate (fructose), as well as Mg, inhibition; FKII was inhibited by neither fructose nor Mg; and FKIII was inhibited by fructose but not by Mg. ATP was the preferred nucleotide donor for all three FKs and FKI showed inhibition by CTP and GTP above 1 mM. All three FKs showed competitive inhibition by ADP. During the maturation of the tomato fruit total FK activity decreased dramatically. There were decreases in activity of all three FKs, nevertheless, all were still observed in the ripe fruit. The two tomato LeFRK genes were expressed in yeast and the gene products were characterized with respect to the distinguishing characteristics of fructose, Mg and nucleotide inhibition. Our results indicate that FKI is the gene product of LeFRK2 and FKII is probably the gene product of LeFRK1.
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Divergent Fructokinase genes are differentially expressed in tomato.
Plant physiology, 1997Co-Authors: Yoshinori Kanayama, Arthur A. Schaffer, Marina Petreikov, Nir Dai, David Granot, Alan B. BennettAbstract:Two cDNA clones (Frk1 and Frk2) encoding Fructokinase (EC 2.7.1.4) were isolated from tomato (Lycopersicon esculentum). The Frk2 cDNA encoded a deduced protein of 328 amino acids that was more than 90% identical with a previously characterized potato (Solanum tuberosum) Fructokinase. In contrast, the Frk1 cDNA encoded a deduced protein of 347 amino acids that shared only 55% amino acid identity with Frk2. Both deduced proteins possessed and ATP-binding motif and putative substrate recognition site sequences identified in bacterial Fructokinases. The Frk1 cDNA was expressed in a mutant yeast (Saccharomyces cerevisiae) line, which lacks the ability to phosphorylate glucose and fructose and is unable to grow on glucose or fructose. Mutant cells expressing Frk1 were complemented to grow on fructose but not glucose, indicating that Frk1 phosphorylates fructose but not glucose, and this activity was verified in extracts of transformed yeast. The mRNA corresponding to Frk2 accumulated to high levels in young, developing tomato fruit, whereas the Frk1 mRNA accumulated to higher levels late in fruit development. The results indicate that Fructokinase in tomato is encoded by two divergent genes, which exhibit a differential pattern of expression during fruit development.
Nir Dai - One of the best experts on this subject based on the ideXlab platform.
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Cloning, expression and characterization of LeFRK3, the fourth tomato (Lycopersicon esculentum Mill.) gene encoding Fructokinase☆
Plant Science, 2004Co-Authors: Marcelo A. German, Arthur A. Schaffer, Marina Petreikov, Israel Asher, Nir Dai, David GranotAbstract:Abstract A full-length cDNA encoding a novel fourth Fructokinase, LeFRK3, was cloned from green tomato (Lycopersicon esculentum Mill.) fruits. The putative protein shares 70, 65.5 and 69% amino acid homology with the three previously identified tomato Fructokinases encoded by LeFRK1, LeFRK2 and LeFRK4, respectively. This fourth Fructokinase has signature patterns of the pfkB family of carbohydrate kinases as well as substrate recognition sites and an ATP-binding domain. Confirmation for its Fructokinase activity was obtained by complementation of triple mutant yeast cells that are unable to phosphorylate or grow on either glucose or fructose as LeFRK3 complemented growth on fructose but not on glucose. Moreover, soluble crude protein extracts prepared from the transformed yeast cells revealed fructose but not glucose phosphorylation activity. In contrast to the LeFRK1 gene product which is inhibited neither by fructose nor by Mg, and to LeFRK2 gene product which is inhibited by both fructose and Mg, the LeFRK3 product is inhibited by fructose but not by Mg. Separation by HPLC-ion exchange chromatography pointed to the gene product of LeFRK3 as the protein responsible for the third peak of Fructokinase activity (FKIII), sharing the same pattern of fructose inhibition previously identified with FKIII in tomato fruits. Mapping of tomato Fructokinases indicated that LeFRK3 is located on chromosome 2, unlike LeFRK1 (chromosome 3), LeFRK2 (chromosome 6), and LeFRK4 (chromosome 10). The relative expression levels of the four known FRK genes in different tomato organs were analyzed by quantitative RT-PCR. LeFRK2 and LeFRK3 are the predominant genes expressed in all organs with LeFRK3 having the highest level of expression in leaves and apices. LeFRK4 is expressed only in stamens. This differential expression patterns combined with the different biochemical characteristics of the four FRK isozymes suggest that each plays a different role in plant development.
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suppression of Fructokinase encoded by lefrk2 in tomato stem inhibits growth and causes wilting of young leaves
Plant Journal, 2003Co-Authors: Marcelo A. German, Arthur A. Schaffer, Marina Petreikov, Nir Dai, Tanya Matsevitz, Rana Hanael, Nirit Bernstein, Marina Ioffe, Yosepha Shahak, David GranotAbstract:Summary Fructokinases catalyze the key step of fructose phosphorylation in plants. LeFRK2, the major Fructokinase-encoding gene in tomato plants, is abundantly expressed in roots, stems, and fruits. To analyze the role of LeFRK2 in plant development, we analyzed transgenic tomato plants with sense and antisense expression of StFRK, the potato homolog of LeFRK2. Increased Fructokinase activity had no effect. However, plants in which LeFRK2 was specifically suppressed, either via antisense suppression or via co-suppression, exhibited growth inhibition and wilting of young leaves at daytime. Grafting experiments indicated that a stem interstock of antisense plants was sufficient to inhibit growth and cause leaf wilting. Stem secondary xylem exhibited particular suppression of LeFRK2 and the area of active xylem, estimated by eosin uptake, was significantly smaller in antisense stem compared to that of wild-type plants. These results suggest that LeFRK2 might be required for proper development of xylem that affected growth and wilting.
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LeFRK4, a novel tomato (Lycopersicon esculentum Mill.) Fructokinase specifically expressed in stamens
Plant Science, 2002Co-Authors: Marcelo A. German, Arthur A. Schaffer, Nir Dai, Inna Chmelnitsky, Irina Sobolev, Yehiam Salts, Rivka Barg, David GranotAbstract:Abstract A full-length cDNA clone encoding a novel Fructokinase, LeFRK4, was isolated from a tomato (Lycopersicon esculentum Mill.) flower cDNA library. The putative protein shares 62.6 and 55.5% amino acid identity with the two known tomato Fructokinases, LeFRK1 and LeFRK2, respectively, and possesses three signature patterns of the pfkB family of carbohydrate kinases, two substrate recognition sites and an ATP-binding domain. The identification of LeFRK4 as a Fructokinase was confirmed by complementation of mutant yeast cells unable to phosphorylate or grow on either glucose or fructose. LeFRK4 complemented growth on fructose but not on glucose. Non-soluble crude protein extracts prepared from the transformed yeast cells exhibited fructose but not glucose phosphorylation activity. Expression analysis demonstrated that LeFRK4 is specifically expressed in stamens. Using quantitative RT-PCR, we examined the relative expression of the three known FRK genes in different tomato organs. LeFRK2, encoding a substrate-inhibited Fructokinase, was by far the predominantly expressed FRK gene in all organs, except for flowers in which it shared the same expression level with LeFRK4. The exclusive expression of LeFRK4 in stamens may point to a specific role of LeFRK4 in pollen development, anthesis, and perhaps pollination.
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LeFRK2, the gene encoding the major Fructokinase in tomato fruits, is not required for starch biosynthesis in developing fruits
Plant Science, 2002Co-Authors: Nir Dai, Arthur A. Schaffer, Marina Petreikov, Marcelo A. German, Tanya Matsevitz, Rana Hanael, Dvora Swartzberg, Yelena Yeselson, David GranotAbstract:Abstract The LeFRK2 gene product has been proposed to play a role in transient starch accumulation in developing tomato (Lycopersicon esculentum) fruits. To analyze the role of LeFRK2 in starch biosynthesis, we produced transgenic tomato plants with sense and antisense expression of StFRK, the potato homologue to LeFRK2. Fruits of homozygous plants expressing sense or antisense StFRK exhibited suppression of LeFRK2, concomitantly with specific elimination of the FKI isozyme, indicating that FKI is the gene product of LeFRK2. The activity of Fructokinase was reduced in antisense and sense homozygous fruits and increased in fruits of sense hemizygous plants. The modified activities of Fructokinase led to small but significant changes both in the steady state levels of sugars and in the level of phosphorylated sugars in fruits. However, fruits lacking FKI had increased rather than decreased starch content. We therefore concluded that LeFRK2 is not required for transient starch accumulation in tomato fruits. Rather, LeFRK2 might have a role in the regulation of sucrose import into tomato fruits.
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Characterization of native and yeast-expressed tomato fruit Fructokinase enzymes
Phytochemistry, 2001Co-Authors: Marina Petreikov, Nir Dai, David Granot, Arthur A. SchafferAbstract:Three Fructokinase isozymes (FKI, FKII, FKIII) were separated from both immature and ripe tomato fruit pericarp. All three isozymes were specific for fructose with undetectable activity towards glucose or mannose. The three isozymes could be distinguished from one another with respect to response to fructose, Mg and nucleotide donor concentrations and this allowed the comparison of the fruit enzymes with the gene products of the two known cloned tomato Fructokinase genes, LeFRK1 and LeFRK2. FKI was characterized by both substrate (fructose), as well as Mg, inhibition; FKII was inhibited by neither fructose nor Mg; and FKIII was inhibited by fructose but not by Mg. ATP was the preferred nucleotide donor for all three FKs and FKI showed inhibition by CTP and GTP above 1 mM. All three FKs showed competitive inhibition by ADP. During the maturation of the tomato fruit total FK activity decreased dramatically. There were decreases in activity of all three FKs, nevertheless, all were still observed in the ripe fruit. The two tomato LeFRK genes were expressed in yeast and the gene products were characterized with respect to the distinguishing characteristics of fructose, Mg and nucleotide inhibition. Our results indicate that FKI is the gene product of LeFRK2 and FKII is probably the gene product of LeFRK1.
Marcelo A. German - One of the best experts on this subject based on the ideXlab platform.
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Cloning, expression and characterization of LeFRK3, the fourth tomato (Lycopersicon esculentum Mill.) gene encoding Fructokinase☆
Plant Science, 2004Co-Authors: Marcelo A. German, Arthur A. Schaffer, Marina Petreikov, Israel Asher, Nir Dai, David GranotAbstract:Abstract A full-length cDNA encoding a novel fourth Fructokinase, LeFRK3, was cloned from green tomato (Lycopersicon esculentum Mill.) fruits. The putative protein shares 70, 65.5 and 69% amino acid homology with the three previously identified tomato Fructokinases encoded by LeFRK1, LeFRK2 and LeFRK4, respectively. This fourth Fructokinase has signature patterns of the pfkB family of carbohydrate kinases as well as substrate recognition sites and an ATP-binding domain. Confirmation for its Fructokinase activity was obtained by complementation of triple mutant yeast cells that are unable to phosphorylate or grow on either glucose or fructose as LeFRK3 complemented growth on fructose but not on glucose. Moreover, soluble crude protein extracts prepared from the transformed yeast cells revealed fructose but not glucose phosphorylation activity. In contrast to the LeFRK1 gene product which is inhibited neither by fructose nor by Mg, and to LeFRK2 gene product which is inhibited by both fructose and Mg, the LeFRK3 product is inhibited by fructose but not by Mg. Separation by HPLC-ion exchange chromatography pointed to the gene product of LeFRK3 as the protein responsible for the third peak of Fructokinase activity (FKIII), sharing the same pattern of fructose inhibition previously identified with FKIII in tomato fruits. Mapping of tomato Fructokinases indicated that LeFRK3 is located on chromosome 2, unlike LeFRK1 (chromosome 3), LeFRK2 (chromosome 6), and LeFRK4 (chromosome 10). The relative expression levels of the four known FRK genes in different tomato organs were analyzed by quantitative RT-PCR. LeFRK2 and LeFRK3 are the predominant genes expressed in all organs with LeFRK3 having the highest level of expression in leaves and apices. LeFRK4 is expressed only in stamens. This differential expression patterns combined with the different biochemical characteristics of the four FRK isozymes suggest that each plays a different role in plant development.
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suppression of Fructokinase encoded by lefrk2 in tomato stem inhibits growth and causes wilting of young leaves
Plant Journal, 2003Co-Authors: Marcelo A. German, Arthur A. Schaffer, Marina Petreikov, Nir Dai, Tanya Matsevitz, Rana Hanael, Nirit Bernstein, Marina Ioffe, Yosepha Shahak, David GranotAbstract:Summary Fructokinases catalyze the key step of fructose phosphorylation in plants. LeFRK2, the major Fructokinase-encoding gene in tomato plants, is abundantly expressed in roots, stems, and fruits. To analyze the role of LeFRK2 in plant development, we analyzed transgenic tomato plants with sense and antisense expression of StFRK, the potato homolog of LeFRK2. Increased Fructokinase activity had no effect. However, plants in which LeFRK2 was specifically suppressed, either via antisense suppression or via co-suppression, exhibited growth inhibition and wilting of young leaves at daytime. Grafting experiments indicated that a stem interstock of antisense plants was sufficient to inhibit growth and cause leaf wilting. Stem secondary xylem exhibited particular suppression of LeFRK2 and the area of active xylem, estimated by eosin uptake, was significantly smaller in antisense stem compared to that of wild-type plants. These results suggest that LeFRK2 might be required for proper development of xylem that affected growth and wilting.
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LeFRK4, a novel tomato (Lycopersicon esculentum Mill.) Fructokinase specifically expressed in stamens
Plant Science, 2002Co-Authors: Marcelo A. German, Arthur A. Schaffer, Nir Dai, Inna Chmelnitsky, Irina Sobolev, Yehiam Salts, Rivka Barg, David GranotAbstract:Abstract A full-length cDNA clone encoding a novel Fructokinase, LeFRK4, was isolated from a tomato (Lycopersicon esculentum Mill.) flower cDNA library. The putative protein shares 62.6 and 55.5% amino acid identity with the two known tomato Fructokinases, LeFRK1 and LeFRK2, respectively, and possesses three signature patterns of the pfkB family of carbohydrate kinases, two substrate recognition sites and an ATP-binding domain. The identification of LeFRK4 as a Fructokinase was confirmed by complementation of mutant yeast cells unable to phosphorylate or grow on either glucose or fructose. LeFRK4 complemented growth on fructose but not on glucose. Non-soluble crude protein extracts prepared from the transformed yeast cells exhibited fructose but not glucose phosphorylation activity. Expression analysis demonstrated that LeFRK4 is specifically expressed in stamens. Using quantitative RT-PCR, we examined the relative expression of the three known FRK genes in different tomato organs. LeFRK2, encoding a substrate-inhibited Fructokinase, was by far the predominantly expressed FRK gene in all organs, except for flowers in which it shared the same expression level with LeFRK4. The exclusive expression of LeFRK4 in stamens may point to a specific role of LeFRK4 in pollen development, anthesis, and perhaps pollination.
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LeFRK2, the gene encoding the major Fructokinase in tomato fruits, is not required for starch biosynthesis in developing fruits
Plant Science, 2002Co-Authors: Nir Dai, Arthur A. Schaffer, Marina Petreikov, Marcelo A. German, Tanya Matsevitz, Rana Hanael, Dvora Swartzberg, Yelena Yeselson, David GranotAbstract:Abstract The LeFRK2 gene product has been proposed to play a role in transient starch accumulation in developing tomato (Lycopersicon esculentum) fruits. To analyze the role of LeFRK2 in starch biosynthesis, we produced transgenic tomato plants with sense and antisense expression of StFRK, the potato homologue to LeFRK2. Fruits of homozygous plants expressing sense or antisense StFRK exhibited suppression of LeFRK2, concomitantly with specific elimination of the FKI isozyme, indicating that FKI is the gene product of LeFRK2. The activity of Fructokinase was reduced in antisense and sense homozygous fruits and increased in fruits of sense hemizygous plants. The modified activities of Fructokinase led to small but significant changes both in the steady state levels of sugars and in the level of phosphorylated sugars in fruits. However, fruits lacking FKI had increased rather than decreased starch content. We therefore concluded that LeFRK2 is not required for transient starch accumulation in tomato fruits. Rather, LeFRK2 might have a role in the regulation of sucrose import into tomato fruits.
