Fruit ripening is a unique biological process only appeared in plant kingdom to yield subsequent generations of viable and competitive progeny. The molecular and cellular processes underlying the development and maturation of fruits have attracted much interest, as the control of fruit ripening is e...
Fruit ripening is a unique biological process only appeared in plant kingdom to yield subsequent generations of viable and competitive progeny. The molecular and cellular processes underlying the development and maturation of fruits have attracted much interest, as the control of fruit ripening is economically important and intimately tied to the human health and diet. Fruit ripening is a complex, genetically programmed process involving biochemical, physiological and structural changes that include degradation of chlorophyll, synthesis of flavor compounds, carotenoid biosynthesis, conversion of starch to sugars, cell wall degradation and fruit softening. These dramatic changes are regulated by both internal and external factors, such as ripening-induced genes, hormones, nutrients, light and temperature. Therefore, the investigation of molecular and biochemical mechanisms regulating fruit ripening are very important to understanding of the fruit ripening process.In the first part of the thesis, results from the characterization of ethylene biosynthesis genes are presented. A wealth of information has been accumulated that plant hormone ethylene is critical for the ripening of climacteric fruits, such as tomato, banana, and apple (Malus domestica Borkh.) that are used for the main plant material in this thesis. In tomato plants, inhibition of ethylene synthesis and its perception completely prevents fruit ripening, demonstrating that ethylene is indeed an essential hormone for ripening. In higher plants, ethylene is generated from the amino acid methionine (Met) via AdoMet (S-adenosyl-L-methionine) and ACC (1-aminocyclopropane-1-carboxylate; Met ? AdoMet ? ACC ? C2H4). The individual reactions of this biosynthetic pathway are catalyzed by AdoMet synthetase, ACC synthase and ACC oxidase, respectively. In vegetative tissues, ACC oxidase is constitutively expressed so that ACC synthase activity is generally regarded as the rate-limiting step for ethylene biosynthesis. In ripening fruit and senescing flower tissues, however, these two unique enzymes are induced during developmental processes and contribute equally to the regulation of ethylene biosynthesis.ACC oxidase requires Fe2+, ascorbate and CO2 as well as substrate ACC, for its enzyme activity. Previous studies of the biochemical mode and catalytic mechanism of ACC oxidase using site-directed mutagenesis of the bacterially expressed protein have suggested that the charged residues near the ligand-binding site are essential for in vitro activity of ACC oxidase. However, no detailed structural information combined with kinetic data to prove this hypothesis has been reported so far. In the chapter I and II of the thesis, we used comparative modeling methods together with site-directed mutagenesis to show the biochemical characteristics of ACC oxidase (MD-ACO1) from apple. The MD-ACO1 protein folds into a compact jelly-roll motif comprised of eight ?\-helices, 12 ?]-strands and several long loops. The active site is well defined as a wide cleft near the C-terminus. The co-substrate, ascorbate molecule is located in cofactor Fe2+-binding pocket, the so-called '2-His-1-carboxylate facial triad'. In addition, our data reveals that Arg-244 and Ser-246 are involved in generating the reaction product during enzyme catalysis and that Lys-296 and Arg-299 residues in the C-terminal helix could be a major stabilizer in the spatial arrangement of reactants. The structure agrees well with the biochemical and site-directed mutagenesis results. The three-dimensional structure together with the steady-state kinetics of both the wild-type and mutant MD-ACO1 proteins reveals how the substrate specificity of MD-ACO1 is involved in the catalytic mechanism, providing insights into the understanding of the fruit ripening process at atomic resolution.ACC synthase gene expression is regulated by more diverse developmental and environmental factors than that of ACC oxidase. In particular, ACC synthase induced by other plant hormones; it is one of the best known hormone interactions in plant biology that auxin enhances the transcriptional activity of a set of ACC synthase gene family members, resulting in increases of ethylene. In Arabidopsis, among the 12 ACC synthase genes identified, the AtACS4 gene is rapidly and specifically activated by auxin within 25 min, whereas expression of AtACS5 is regulated by cytokinin by a post-transcriptional mechanism. In addition to auxin and cytokinin, brassinosteroid (BR), a growth-promoting natural compound with similar structure to animal steroid hormones, has been shown to induce ethylene production. In etiolated mung bean seedlings, BR acts synergistically with auxin to stimulate ethylene synthesis by inducing ACC synthase genes. However, the molecular mechanism of BR-induced ethylene production has not been addressed in Arabidopsis plants. Chapter III consists of the results for identifying the molecular relationship between auxin and BR to induce the ethylene production in Arabidopsis seedlings. We show that BR specifically enhanced the expression of AtACS4, which encodes an auxin-responsive ACC synthase 4, by a distinct temporal induction mechanism compared with that of IAA in etiolated Arabidopsis seedlings. This BR induction of AtACS4 was undetectable in the light-grown seedlings. In addition, BR failed to activate the AtACS4 gene in auxin-resistant1 (axr1-3) and auxin-resistant2 (axr2-1), both of which are auxin-resistant mutants. Thus, it appears that there is a possible regulatory link between light, auxin and BR to control ethylene synthesis in Arabidopsis young seedlings. Analysis of transgenic Arabidopsis plants harboring AtACS4::GUS fusion revealed the AtACS4 promoter-driven GUS activity in the highly elongating zone of the hypocotyls in response to BR treatment. Furthermore, Arabidopsis plants homozygous for the T-DNA insertion in the AtACS4 gene exhibited longer hypocotyls and roots than those of control seedlings. Taken together, these results suggest that the BR-induced ethylene production may participate in the elongation growth response in early seedling development of Arabidopsis.Another part of the thesis is the study on functional genomics and bioengineering related to fruit ripening of Fuji apple (Malus domestica Borkh. cv. Fuji). Although the dramatic changes that plants undergo during ripening are caused by the activation of a number of genes, including ethylene biosynthesis genes that are studied above part of the thesis, there is a very limited amount of genome-wide research resource for the apple, which is recognized as the major horticultural fruit crop. To investigate the molecular mechanism involved in the regulation of fruit ripening in Fuji apple, we constructed a cDNA library and EST database using poly (A)+ RNA from full-ripened Fuji apple fruits. Results from clustering of 1,117 ESTs by homology based gene functions revealed that the group of genes involved in metabolism (21.5%), biotic/abiotic stress (12.1%) and protein synthesis/degradation (11.0%) are present in a large proportion of expressed genes at full-ripening stage. To identify the ripening-specific genes that are induced only at stage IV, we investigated the expression profiles of these ESTs using apple fruit cDNA microarray. The result from the analysis of cDNA microarray showed that 159 cDNA clones are specifically expressed in full-ripened apple fruits. Like chitinase, AUX/IAA and MADS box protein, a lot of genes known as fruit-specific or ripening-specific gene in other plants were included in apple fruit-ripening specific genes. In particular, 6 genes encoding the enzymes that participate in ubiquitin-mediated protein degradation pathway were specifically expressed at full-ripened stage of apple fruits. From these results, we suggest the basis of further investigation for elucidating the molecular mechanisms involved in the regulation of Fuji apple fruit ripening.In addition to elucidating the molecular and biochemical events that determine fruit ripening, the modification of gene expression offers the potential to improve fruit quality by altering biochemical pathways that contribute to flavor, color, and aroma or by increasing shelf-life and/or regulating the initiation and rate of the ripening process. Tocopherols (vitamin E) that are an essential fat-soluble vitamin and synthesized only by photosynthetic organisms function as a free radical scavenger, thereby effectively inhibiting lipid oxidation in mammals. Although the exact function of tocopherols in plants is not well defined, the generation of substantial increases in amounts of tocopherols by genetic engineering may lead to increased resistance of plant tissues to oxidative stresses. Therefore, the genetic enhancement of tocotrienol content by the overexpression of homogentisate phytyltransferase (HPT) gene in fruit tissue will provide improved antioxidant capacity to transgenic fruits, which have improved productivity, shelf life, and oxidative stability. In the last chapter, for enhancing the amount of tocopherol in fruit we isolated and charaterized MdHPT1 in Fuji apple. The MdHPT1 encoded HPT, which were confimed by complementation experiment with the HPT null mutant of cyanobacteria Synechocystis sp. PCC6803 (slr1736). We also expressed MdHPT1 in Arabidopsis ectopically, and are investigating the tocopherol content of trangenic Arabidopsis. Because the overexpression of HPT gene in Arabidopsis and corn resulted in a strong increase in tocopherol synthesis of transgenic seeds and leaves, we expect so in the MdHPT1 overexpressed Arabidopsis. After confirming in vivo function of MdHPT1 from above results, we will express MdHPT1 in tomato and apple using fruit-ripening specific promoter. In particular, because Fuji apple MdHPT1 mRNA and tocopherol level were low in both immature and ripened fruits, we expect substantial increases in amount of tocophe...
Fruit ripening is a unique biological process only appeared in plant kingdom to yield subsequent generations of viable and competitive progeny. The molecular and cellular processes underlying the development and maturation of fruits have attracted much interest, as the control of fruit ripening is economically important and intimately tied to the human health and diet. Fruit ripening is a complex, genetically programmed process involving biochemical, physiological and structural changes that include degradation of chlorophyll, synthesis of flavor compounds, carotenoid biosynthesis, conversion of starch to sugars, cell wall degradation and fruit softening. These dramatic changes are regulated by both internal and external factors, such as ripening-induced genes, hormones, nutrients, light and temperature. Therefore, the investigation of molecular and biochemical mechanisms regulating fruit ripening are very important to understanding of the fruit ripening process.In the first part of the thesis, results from the characterization of ethylene biosynthesis genes are presented. A wealth of information has been accumulated that plant hormone ethylene is critical for the ripening of climacteric fruits, such as tomato, banana, and apple (Malus domestica Borkh.) that are used for the main plant material in this thesis. In tomato plants, inhibition of ethylene synthesis and its perception completely prevents fruit ripening, demonstrating that ethylene is indeed an essential hormone for ripening. In higher plants, ethylene is generated from the amino acid methionine (Met) via AdoMet (S-adenosyl-L-methionine) and ACC (1-aminocyclopropane-1-carboxylate; Met ? AdoMet ? ACC ? C2H4). The individual reactions of this biosynthetic pathway are catalyzed by AdoMet synthetase, ACC synthase and ACC oxidase, respectively. In vegetative tissues, ACC oxidase is constitutively expressed so that ACC synthase activity is generally regarded as the rate-limiting step for ethylene biosynthesis. In ripening fruit and senescing flower tissues, however, these two unique enzymes are induced during developmental processes and contribute equally to the regulation of ethylene biosynthesis.ACC oxidase requires Fe2+, ascorbate and CO2 as well as substrate ACC, for its enzyme activity. Previous studies of the biochemical mode and catalytic mechanism of ACC oxidase using site-directed mutagenesis of the bacterially expressed protein have suggested that the charged residues near the ligand-binding site are essential for in vitro activity of ACC oxidase. However, no detailed structural information combined with kinetic data to prove this hypothesis has been reported so far. In the chapter I and II of the thesis, we used comparative modeling methods together with site-directed mutagenesis to show the biochemical characteristics of ACC oxidase (MD-ACO1) from apple. The MD-ACO1 protein folds into a compact jelly-roll motif comprised of eight ?\-helices, 12 ?]-strands and several long loops. The active site is well defined as a wide cleft near the C-terminus. The co-substrate, ascorbate molecule is located in cofactor Fe2+-binding pocket, the so-called '2-His-1-carboxylate facial triad'. In addition, our data reveals that Arg-244 and Ser-246 are involved in generating the reaction product during enzyme catalysis and that Lys-296 and Arg-299 residues in the C-terminal helix could be a major stabilizer in the spatial arrangement of reactants. The structure agrees well with the biochemical and site-directed mutagenesis results. The three-dimensional structure together with the steady-state kinetics of both the wild-type and mutant MD-ACO1 proteins reveals how the substrate specificity of MD-ACO1 is involved in the catalytic mechanism, providing insights into the understanding of the fruit ripening process at atomic resolution.ACC synthase gene expression is regulated by more diverse developmental and environmental factors than that of ACC oxidase. In particular, ACC synthase induced by other plant hormones; it is one of the best known hormone interactions in plant biology that auxin enhances the transcriptional activity of a set of ACC synthase gene family members, resulting in increases of ethylene. In Arabidopsis, among the 12 ACC synthase genes identified, the AtACS4 gene is rapidly and specifically activated by auxin within 25 min, whereas expression of AtACS5 is regulated by cytokinin by a post-transcriptional mechanism. In addition to auxin and cytokinin, brassinosteroid (BR), a growth-promoting natural compound with similar structure to animal steroid hormones, has been shown to induce ethylene production. In etiolated mung bean seedlings, BR acts synergistically with auxin to stimulate ethylene synthesis by inducing ACC synthase genes. However, the molecular mechanism of BR-induced ethylene production has not been addressed in Arabidopsis plants. Chapter III consists of the results for identifying the molecular relationship between auxin and BR to induce the ethylene production in Arabidopsis seedlings. We show that BR specifically enhanced the expression of AtACS4, which encodes an auxin-responsive ACC synthase 4, by a distinct temporal induction mechanism compared with that of IAA in etiolated Arabidopsis seedlings. This BR induction of AtACS4 was undetectable in the light-grown seedlings. In addition, BR failed to activate the AtACS4 gene in auxin-resistant1 (axr1-3) and auxin-resistant2 (axr2-1), both of which are auxin-resistant mutants. Thus, it appears that there is a possible regulatory link between light, auxin and BR to control ethylene synthesis in Arabidopsis young seedlings. Analysis of transgenic Arabidopsis plants harboring AtACS4::GUS fusion revealed the AtACS4 promoter-driven GUS activity in the highly elongating zone of the hypocotyls in response to BR treatment. Furthermore, Arabidopsis plants homozygous for the T-DNA insertion in the AtACS4 gene exhibited longer hypocotyls and roots than those of control seedlings. Taken together, these results suggest that the BR-induced ethylene production may participate in the elongation growth response in early seedling development of Arabidopsis.Another part of the thesis is the study on functional genomics and bioengineering related to fruit ripening of Fuji apple (Malus domestica Borkh. cv. Fuji). Although the dramatic changes that plants undergo during ripening are caused by the activation of a number of genes, including ethylene biosynthesis genes that are studied above part of the thesis, there is a very limited amount of genome-wide research resource for the apple, which is recognized as the major horticultural fruit crop. To investigate the molecular mechanism involved in the regulation of fruit ripening in Fuji apple, we constructed a cDNA library and EST database using poly (A)+ RNA from full-ripened Fuji apple fruits. Results from clustering of 1,117 ESTs by homology based gene functions revealed that the group of genes involved in metabolism (21.5%), biotic/abiotic stress (12.1%) and protein synthesis/degradation (11.0%) are present in a large proportion of expressed genes at full-ripening stage. To identify the ripening-specific genes that are induced only at stage IV, we investigated the expression profiles of these ESTs using apple fruit cDNA microarray. The result from the analysis of cDNA microarray showed that 159 cDNA clones are specifically expressed in full-ripened apple fruits. Like chitinase, AUX/IAA and MADS box protein, a lot of genes known as fruit-specific or ripening-specific gene in other plants were included in apple fruit-ripening specific genes. In particular, 6 genes encoding the enzymes that participate in ubiquitin-mediated protein degradation pathway were specifically expressed at full-ripened stage of apple fruits. From these results, we suggest the basis of further investigation for elucidating the molecular mechanisms involved in the regulation of Fuji apple fruit ripening.In addition to elucidating the molecular and biochemical events that determine fruit ripening, the modification of gene expression offers the potential to improve fruit quality by altering biochemical pathways that contribute to flavor, color, and aroma or by increasing shelf-life and/or regulating the initiation and rate of the ripening process. Tocopherols (vitamin E) that are an essential fat-soluble vitamin and synthesized only by photosynthetic organisms function as a free radical scavenger, thereby effectively inhibiting lipid oxidation in mammals. Although the exact function of tocopherols in plants is not well defined, the generation of substantial increases in amounts of tocopherols by genetic engineering may lead to increased resistance of plant tissues to oxidative stresses. Therefore, the genetic enhancement of tocotrienol content by the overexpression of homogentisate phytyltransferase (HPT) gene in fruit tissue will provide improved antioxidant capacity to transgenic fruits, which have improved productivity, shelf life, and oxidative stability. In the last chapter, for enhancing the amount of tocopherol in fruit we isolated and charaterized MdHPT1 in Fuji apple. The MdHPT1 encoded HPT, which were confimed by complementation experiment with the HPT null mutant of cyanobacteria Synechocystis sp. PCC6803 (slr1736). We also expressed MdHPT1 in Arabidopsis ectopically, and are investigating the tocopherol content of trangenic Arabidopsis. Because the overexpression of HPT gene in Arabidopsis and corn resulted in a strong increase in tocopherol synthesis of transgenic seeds and leaves, we expect so in the MdHPT1 overexpressed Arabidopsis. After confirming in vivo function of MdHPT1 from above results, we will express MdHPT1 in tomato and apple using fruit-ripening specific promoter. In particular, because Fuji apple MdHPT1 mRNA and tocopherol level were low in both immature and ripened fruits, we expect substantial increases in amount of tocophe...
주제어
#과일 성숙
#생물 공학
#비교 구조 예측
#에틸렌
#옥신
#브라시노스테로이드
#항산화
#활성 산소
#토코페롤
#비타민 E
#과일 성숙 특이적 유전자
#사과
#애기장대
#시아노박테리아
#fruit ripening
#fruit quality
#Expressed Sequence Tags
#EST
#microarray
#bioengineering
#comparative modeling
#ethylene
#auxin
#brassinosteroid
#BR
#antioxidant
#oxidative stress
#tocopherols
#vitamin E
#ACC oxidase
#ACC synthase
#homogentisate phytyltransferase
#HPT
#MD-ACO1
#AtACS4
#MdHPT1
#slr1736
#ripening-specific genes
#apple (Malus domestica Borkh.)
#Arabidopsis thaliana
#cyanobacteria Synechocystis sp. PCC6803
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