Traditional methods like pruning and breeding have historically been used in crop production to divert photoassimilates to harvested organs, but molecular biotechnology is now poised to significantly increase yield by manipulating resource partitioning. It was hypothesized that metabolic engineering in targeted sink tissues can favor resource partitioning to increase harvest. Raffinose Family Oligosaccharides (RFOs) are naturally occurring oligosaccharides that are widespread in plants and are responsible for carbon transport, storage and protection against cold and drought stress. Transgenic plants (GRS47, GRS63) were engineered to generate and transport more RFOs through the phloem than the wild type plants. The transgenic lines produced more RFOs and the RFOs were also detected in their phloem exudates. But the 14CO2 labeling and subsequent thin layer chromatography analysis showed that the RFOs were most likely sequestered in an inactive pool and accumulate over time. Crossing GRS47 and GRS63 lines with MIPS1 plants (that produces more myo-inositol, a substrate in the RFO biosynthetic pathway) did not significantly increase the RFOs in the crossed lines. For future manipulation of RFO degradation in sink organs, the roles of the endogenous α-galactosidases were analyzed. The alkaline α-galactosidases (AtSIP1 and AtSIP2 in Arabidopsis) are most likely responsible for digesting RFOs in the cytoplasm and may influence the ability to manipulate RFO levels in engineered plants. Atsip1/2 (AtSIP1/AtSIP2 double-knockout plants) were generated and phenotypically characterized based on seed germination patterns, flowering time, and sugar content to observe the impact on RFO sugar levels. The observations and analysis from these lines provide a basis for further insight in the manipulation of resource allocation between source and sink tissues in plants for future research.

To increase the global acceptability of chickpea by improving its nutritional quality, seed RFO (Raffinose Family Oligosaccharides) concentration needs to be reduced without affecting their role during seed development and positive impact on human health. To achieve this objective, the key regulating step(s) of RFO biosynthesis needs to be identified. The three main objectives of the thesis were: (1) to optimize an analytical method to determine soluble sugars concentration in chickpea seeds including RFO, (2) to determine chickpea genotypes with contrasting seed RFO concentration, and (3) to optimize and validate RFO biosynthetic enzyme activity assays. These three objectives of the thesis provided basis of the fourth objective. For the first objective, a modified HPAEC-PAD (High performance anion exchange chromatography with pulsed amperometric detector) based gradient approach was optimized to study the concentration and composition of soluble sugars in chickpea seeds. The optimized method separated all the soluble sugars within 20 min of run time with higher accuracy, sensitivity and precision compared to previously reported methods. Therefore, the optimized method was utilized to study the natural variation in RFO concentration of 171 chickpea genotypes. Sucrose (0.60 - 3.59 g/100 g) and stachyose (0.18 − 2.38 g/100 g) were predominant among soluble sugars and RFO, respectively. Analysis of variance revealed a significant impact (P ≤ 0.001) of genotype (G), environment (E), and their interaction (G×E) on seed RFO concentration in chickpea. A significant positive correlation was observed between substrate and product concentration in RFO biosynthesis. Raffinose, stachyose and verbascose showed moderate broad sense heritability (0.25 − 0.56) suggesting the quantitative nature of the RFO trait in chickpea seeds. Desi (ICC 1163, ICC 1471, ICC 9562, ICCV 07115, ICCV 07116 and ICCV 07117) and kabuli (ICC 5270, ICC 10674, ICC 16216, ICC 16528, ICCV 3 and ICCV 91302) chickpea genotypes with high and low RFO concentrations (high RFO genotypes are underlined) were identified. RFO biosynthetic enzymes activities were optimized for substrate and protein concentration, temperature (25 °C), time (10 min for galactinol synthase and 60 min for other biosynthetic enzymes) and pH (7.0). These assays were validated at different seed developmental stages of two released varieties: CDC Vanguard and CDC Frontier. Simultaneously, RFO accumulation at different seed developmental stages was also studied. During 18 - 38 DAF (days after flowering), about a 75 % decrease in seed moisture was observed coinciding with the accumulation of RFO providing desiccation tolerance to maturing seeds. The initial substrates viz. myo-inositol and sucrose were observed throughout seed development process having maximum accumulation at 18 - 20 (0.50 – 0.57 g/100 g) and 20 - 22 (9.94 - 11.17 g/100 g) DAF that decreased afterwards supporting the biosynthesis of galactinol and raffinose, respectively. Galactinol is considered as the universal galactosyl donor, it showed the highest concentration at 30 DAF and this was later utilized for increased RFO accumulation till 36 DAF. Activity of RFO biosynthetic enzymes was observed 2 - 6 days prior to first detection of their corresponding products whereas the highest activities were determined 2 - 4 days prior to maximum accumulation of RFO. However, maximum GS (galactinol synthase) activity was observed at 36 DAF but this did not correspond to amount of galactinol accumulation in seeds. This indicated that galactinol was synthesized in higher amount even after 30 DAF but directed towards RFO biosynthesis thus could not necessarily accumulate in seeds. A galactinol independent pathway was also found operative in chickpea seeds. These results suggested that substrate concentration and GS activity might be the possible factors regulating seed RFO concentration in chickpea. The fourth objective utilized the information, material and methods from the previous three objectives. Chickpea genotypes with contrasting RFO concentration were compared for seed size and weight, germination capacity and RFO biosynthesis (accumulation and biosynthetic enzymes activities during seed development). Sucrose concentration showed a significant positive (r = 0.728, P ≤ 0.05) correlation with seed size/weight. RFO concentration was a facilitator of seed germination rather than regulating factor. Higher accumulation of myo-inositol and sucrose in high RFO genotypes during early seed developmental stages suggested that initial substrates concentrations may influence seed RFO concentration. High RFO genotypes expressed about 2 - 3 fold higher activity for all RFO biosynthetic enzymes compared to those with low RFO concentration. The enzyme activity data corresponded with the accumulation of individual RFO during chickpea seed development. In conclusion, regulating galactinol synthase activity is a potential strategy to reduce seed RFO concentration in chickpea. The present study can be extended to study RFO biosynthesis at the transcript level and the influence of RFO biosynthetic enzymes on seed size and weight, germination, RFO concentration, yield, and stress tolerance.

Raffinose family oligosaccharides (RFO) are major soluble carbohydrates of lentil (Lens culinaris Medik) seeds. When consumed by humans, RFO pass indigested through upper digestive tract as ¦Á-galactosidase enzyme required for RFO breakdown is not produced in humans. Consumption of lentils with high concentrations of RFO result in stomach discomfort, bloating and diarrhea due to bacterial fermentation of RFO in large intestine. This has lead to a relatively low use of lentils for human consumption. RFO are therefore considered anti-nutritional factors and development of lentil cultivars with reduced RFO concentrations is desired to improve lentil quality and consumption. To explore the possibility to develop lentil cultivars for low-RFO concentration, heritability of RFO trait and influence of environmental conditions on RFO must be known. In addition, RFO biosynthesis and accumulation in lentil seeds must be understood. However, very limited information is available on the above mentioned aspects of RFO in general and in lentil in particular. Therefore, the objectives of this study were: (1) to evaluate natural variation in RFO concentration and composition in commonly grown lentil cultivars and to determine the correlation between RFO concentration and other important seed constituents, (2) to investigate heritability and effect of environment on concentration and composition of lentil seed soluble carbohydrates, (3) to assess natural variation and diversity in RFO concentration in the genus Lens, (4) and to evaluate the association between galactinol synthase activity and accumulation of RFO in lentil seeds. Analysis of 22 lentil genotypes revealed significant (P ¡Ü 0.05) variation in total starch, amyolse, protein, total RFO and seed weight and seed colour. Stachyose was the major RFO in all lentil genotypes followed by raffinose and verbascose. A significant (P ¡Ü 0.05) inverse correlation was found between RFO and amylose concentration (r = -0.34); whereas RFO concentration and thousand seed weight correlated positively (r = 0.35). The analyses of variance of eleven cultivars grown at ten different environments showed that cultivar, environment and their interaction had significant effects on sugar concentration in lentil seeds. The high broad sense heritability of RFO (h2= 0.85) indicated that RFO concentration in lentil seeds is highly heritable and thus amenable to genetic improvement. An extensive evaluation of domesticated and wild species and subspecies of the genus Lens revealed significant (P ¡Ü 0.05) variation and diversity in RFO concentration and composition of individual oligosaccharides. Higher Shannon-Weaver diversity indices (SDI) for total RFO, raffinose and verbascose traits were observed in wild lentils compared to domesticated genotypes. Lens ervoides genotypes and some wild genotypes contained almost half the RFO concentration of cultivated lentils and therefore, wild genotypes may be useful for developing low-RFO lines. Higher verbascose and lower stachyose concentration was found in Lens ervoides genotypes, whereas higher raffinose and lower verbascose concentration is found in Lens nigricans genotypes. Study of galactinol synthase activity in developing seeds with varying RFO concentration showed no clear association between galactinol synthase activity and RFO concentration. The sucrose and galactinol concentration of developing seeds were also not associated with total RFO concentration of lentil seeds. This finding suggests a non-regulatory role of galactinol synthase in RFO biosynthetic pathway in lentil seeds. Together, all these findings are not only significant to devise strategies to develop lentil cultivars with reduced RFO concentration but also for understanding RFO biosynthesis in lentil seeds.

Raffinose family oligosaccharides (RFO) are major soluble carbohydrates of lentil (Lens culinaris Medik) seeds. When consumed by humans, RFO pass indigested through upper digestive tract as ¦Á-galactosidase enzyme required for RFO breakdown is not produced in humans. Consumption of lentils with high concentrations of RFO result in stomach discomfort, bloating and diarrhea due to bacterial fermentation of RFO in large intestine. This has lead to a relatively low use of lentils for human consumption. RFO are therefore considered anti-nutritional factors and development of lentil cultivars with reduced RFO concentrations is desired to improve lentil quality and consumption. To explore the possibility to develop lentil cultivars for low-RFO concentration, heritability of RFO trait and influence of environmental conditions on RFO must be known. In addition, RFO biosynthesis and accumulation in lentil seeds must be understood. However, very limited information is available on the above mentioned aspects of RFO in general and in lentil in particular. Therefore, the objectives of this study were: (1) to evaluate natural variation in RFO concentration and composition in commonly grown lentil cultivars and to determine the correlation between RFO concentration and other important seed constituents, (2) to investigate heritability and effect of environment on concentration and composition of lentil seed soluble carbohydrates, (3) to assess natural variation and diversity in RFO concentration in the genus Lens, (4) and to evaluate the association between galactinol synthase activity and accumulation of RFO in lentil seeds. Analysis of 22 lentil genotypes revealed significant (P ¡Ü 0.05) variation in total starch, amyolse, protein, total RFO and seed weight and seed colour. Stachyose was the major RFO in all lentil genotypes followed by raffinose and verbascose. A significant (P ¡Ü 0.05) inverse correlation was found between RFO and amylose concentration (r = -0.34); whereas RFO concentration and thousand seed weight correlated positively (r = 0.35). The analyses of variance of eleven cultivars grown at ten different environments showed that cultivar, environment and their interaction had significant effects on sugar concentration in lentil seeds. The high broad sense heritability of RFO (h2= 0.85) indicated that RFO concentration in lentil seeds is highly heritable and thus amenable to genetic improvement. An extensive evaluation of domesticated and wild species and subspecies of the genus Lens revealed significant (P ¡Ü 0.05) variation and diversity in RFO concentration and composition of individual oligosaccharides. Higher Shannon-Weaver diversity indices (SDI) for total RFO, raffinose and verbascose traits were observed in wild lentils compared to domesticated genotypes. Lens ervoides genotypes and some wild genotypes contained almost half the RFO concentration of cultivated lentils and therefore, wild genotypes may be useful for developing low-RFO lines. Higher verbascose and lower stachyose concentration was found in Lens ervoides genotypes, whereas higher raffinose and lower verbascose concentration is found in Lens nigricans genotypes. Study of galactinol synthase activity in developing seeds with varying RFO concentration showed no clear association between galactinol synthase activity and RFO concentration. The sucrose and galactinol concentration of developing seeds were also not associated with total RFO concentration of lentil seeds. This finding suggests a non-regulatory role of galactinol synthase in RFO biosynthetic pathway in lentil seeds. Together, all these findings are not only significant to devise strategies to develop lentil cultivars with reduced RFO concentration but also for understanding RFO biosynthesis in lentil seeds.

Des semis précoces permettent d'allonger les cycles de culture du pois protéagineux et d'éviter les stress hydriques et thermiques tardifs, ce qui contribue à la maîtrise des variations de rendement. Les objectifs de ce travail sont 1) d'étudier le comportement de différents génotypes de pois au cours de la germination en réponse à la température ; 2) de déterminer l'impact des vitesses de dessiccation à température élevée sur les caractéristiques des graines, leur germination et leur conservation ; 3) d'explorer la reprise du métabolisme énergétique et la mobilisation des sucres solubles au cours de la germination et de déterminer l'impact d'une vitesse de dessiccation rapide après la phase de remplissage et des basses températures pendant la germination. Les pois de printemps Baccara et Térèse et le pois d'hiver Champagne présentent une température de base identique et très basse de -1,1°C. Le génotype Champagne a une vitesse de germination plus rapide que les deux autres génotypes, des basses températures jusqu'à l'optimum. Les vitesses de dessiccation élevées ont peu d'impact sur la germination mais affectent fortement l'aptitude à la conservation des graines, mesurée après détérioration contrôlée. Cela est associé à une augmentation de la conductivité traduisant des dégâts cellulaires, en particulier au niveau des membranes. La vitesse élevée de germination de Champagne s'explique en partie par une grande vitesse d'imbibition, permettant une respiration plus forte. Une corrélation entre la consommation en oxygène et la production d'ATP a été trouvée. Champagne présente également une capacité de la voie alternative, une utilisation d'ATP et de sucres solubles plus élevées que les génotypes de printemps, Baccara et Térèse. L'utilisation des sucres solubles des axes embryonnaires, après mobilisation des RFO, est bien corrélée avec la vitesse de germination. De fortes vitesses de dessiccation entrainent une augmentation de la respiration qui serait à relier à la mise en place de mécanismes de réparation. Les basses températures retardent le processus de germination et de reprise du métabolisme par leur action sur les systèmes enzymatiques, mais lorsque l'échelle de temps est exprimée en temps thermique, le retard disparaît. Cependant, la respiration du génotype Champagne est moins affectée par les basses températures. Les résultats obtenus ont permis de proposer un schéma global résumant les étapes clés permettant une vitesse de germination rapide et un modèlesur le rôle de l'adénylate kinase et de la phosphorylation oxydative mitochondriale dans la régulation du métabolisme en lien avec l'anhydrobiose.

Les agrobactéries établissent des relations à long terme avec les plantes et ce, dans deux styles de vie différents, rhizosphérique et pathogène (galle du collet). Dans ce mode de vie, les bactéries modifient génétiquement leur hôte et se créent ainsi une niche écologique spécifique (tumeur). La transition entre les deux styles de vie est déclenchée par la perception de signaux végétaux, parmi lesquels des acides hydroxycinnamiques (HCAs) comme l’acide férulique. Or dans l’espèce Agrobacterium fabrum, des gènes spécifiques permettent la dégradation des HCAs. Nous avons émis l’hypothèse que cette dégradation était un signal de proximité de la plante et influençait alors des fonctions importantes pour l’interaction avec celle-ci. Nous avons caractérisé la régulation de la dégradation des HCAs, évalué son rôle dans la valeur sélective d’A. fabrum, et suggéré son importance dans la transition entre les styles de vie rhizosphérique et pathogène. Nous avons montré que la dégradation des HCAs module le métabolisme carboné bactérien, notamment l’utilisation d’acide aminés et d’oligosaccharides de la famille du raffinose. Nous avons caractérisé la protéine MelB qui permet l’import de ces sucres, du mélibiose et du galactinol. Leur utilisation est importante pour la colonisation des plantes dès la germination. L’analyse de l’expression des gènes et du métabolisme bactérien en présence d'un composé signal de la plante, nous a révélé de nouveaux déterminants importants pour l’écologie de ce phytopathogène, notamment des facteurs de transcription. En outre, cette analyse a confirmé l’importance des échanges cellulaires et de déterminants impliqués dans la compétition bactérienne / Agrobacterium establish long term interactions with plants, either in a rhizosphere or pathogenic lifestyle. Pathogenic agrobacteria are causing the crown gall disease by genetically modifying the plant cells host, thus creating a specific ecological niche (tumor). The transition from the rhizosphere to the pathogenic lifestyle is triggered by bacterial perception of plant-derived signals, including hydroxycinnamic acids (HCAs) such as ferulic acid. However, A. fabrum strains have species-specific genes that allow HCAs degradation.We hypothesized that in A. fabrum, the degradation of the HCAs is perceived as a plant signal which influences important functions involved in the interaction with plants. We characterized the regulation of HCAs degradation, evaluated its role in the fitness of A. fabrum, and suggested its importance for the transition between the rhizosphere and pathogenic lifestyles. Then, we showed that the degradation of HCAs modulates carbon metabolism, such as the use of amino acids and sugars belonging to the raffinose family oligosaccharides (RFO). We have demonstrated that besides these sugars, the MelB protein allows the import melibiose and galactinol. Their use is important for plant colonization, since seed germination. The analyzes of gene expression and bacterial metabolism in the presence of a plant signal compound, revealed new determinants important for A. fabrum ecology, including transcription factors. In addition, it confirmed the importance of cellular exchanges and bacterial competition for Agrobacterium fitness in planta

2005年「大西洋鮪類資源保育委員會」(International Commission for the Conservation of Atlantic Tunas, ICCAT)第十九屆年會將台灣2006年在大西洋大目鮪(Bigeye Tuna)的漁獲配額自14,900公噸削減為4,600公噸的決定，即05-02號建議(Recommendation by ICCAT Regarding Control of Chinese Taipei’s Atlantic Bigeye Tuna Fishery [05-02] in 2005)，除反應出漁業資源日益稀少和各國竸爭激烈的事實外，也顯示了國人海洋漁業生態保育的觀念有待加強。 / 本論文乃以ICCAT 05-02號建議作為個案研究，對高度洄游魚類種群(highly migratory species)之養護與管理體系為一整體檢視，探討該號建議所呈現之生態保育問題。內容側重於法規範面之探討，並就高度洄游魚類種群之定義、生態習性、種類、及資源使用狀況為一基本介紹。主要探討三大議題：(1)國際規範體系架構；(2)養護與管理高度洄游魚類之現況；(3)當前面臨之問題與挑戰。 / 全球海洋資源面臨的人為挑戰模式均同，本文焦點雖在高度洄游魚類種群之探討，但也反應出其他種類漁資源在保育上所面臨的共同問題，只不過因高度洄游魚類生態上的特殊性，使其整體養護與管理過程更能突顯出國際漁業治理的本質─國際合作的必要性。但若從另一角度分析漁業管理制度發生改變的原因，則將突顯出國際漁業治理的另一項本質，即國家利益的追求，事實上這也是所有國際活動的基本特徵。因此，以專業性的角度來分析區域漁業組織運作的同時，不能忽略國際政治因素的牽絆，國家漁業經濟利益透過國際政治上的實力對比在區域漁業組織的運作過程中彰顯出來。 / 本論文章節安排分為六章，首先概略介紹「高度洄游魚類」之意義、特性及資源使用狀態(第一章)，爾後簡介現行有關高度洄游魚類之國際規範體系(第二章)，接著以ICCAT為案例，使讀者明白當前國際間透過區域漁業組織來施行漁資源管理的實際運作情形(第三章)，透過05-02號建議讓讀者了解區域漁業組織如何敦促成員國遵守區域漁業規範，落實其養護與管理高度洄游魚類之國際任務(第四章)，並探討ICCAT05-02號建議所反應出來的制度面或執行面之問題(第五章)。最後綜合各章節內容，針對高度洄游魚類之整體規範體系架構為一觀察反省，歸結出本議題核心所在，即追求國家利益前提下的國際合作(第六章)。 / 國際間對於高度洄游魚類種群的保育工程已然啟動，國家願意讓渡出更多一些的權力由國際專門性機構來主導，並在一定規範架構下進行國際合作，這是值得肯定的發展趨勢，並可觀樂期待國際間對於生態環境議題將給予更多的關注，落實資源永續經營的理念。 / On 19th regular meeting of the International Commission for the Conservation of Atlantic Tunas (ICCAT), the commission decided to cut down Taiwan’s quota of Bigeye tuna in Atlantic Ocean form 14,900 tonnage to 4,900, that is Recommendation by ICCAT Regarding Control of Chinese Taipei’s Atlantic Bigeye Tuna Fishery [05-02] in 2005. This case reflects the fact that fishery resource is coming shorter and States compete harshly. It shows that our concept of conservation ecology leaves much space to improve as well. / This thesis is a case study based on Recommendation 05-02 by ICCAT. Here try to inspect thoroughly the conservation and management system of highly migratory species, so as to discuss the problems of eco-conservation reflecting from that Recommendation. Contexts focus on the regulation aspect, and briefly introduce the definition, ecologic habitual behavior, kinds, and condition of resource consumption of highly migratory species. The three main topics to discuss are: (1) international regulation framework; (2) current status of conservation and management of highly migratory species; (3) the problems and challenges confront with nowadays. / The challenges to ocean resource caused by human activity are all the same. This thesis is about highly migratory species, but also reflects the common problem of other fish species in ecological protection respect. Merely because of the ecological particularity, the whole process of conservation and management of highly migratory species could better show the key ingredient of international fishery governance, which is international cooperation. From the other angle, the change of fishery management system mirrors the nature of international fishery management, that is pursuing of national interest. Actually, the later is fundamental character of all kinds of international interaction. Therefore, when analyzing the function of regional fishery organization, we could not ignore the international political fact. National economic fishery interest manifests through relative international political power in fishery management organizations. / There are six chapters in this thesis. First, to take a quick glance at what “highly migratory species” is and its character and resource status (chapter 1). And then turn to the introduction of current international regulation system of highly migratory species (chapter 2). Following is a case study based on ICCAT, to inform readers how Regional Fishery Organizations function actually (chapter 3). Through Recommendation 05-02 by ICCAT, we could take a look how Regional Fishery Organizations urge States to meet their commission of conserving and managing highly migratory species (chapter 4). And then go to a discussion of institutional and enforcement problems reflect from Recommendation 05-02 by ICCAT (chapter 5). Finally, we take a entirely observation and introspection from the foregoing chapters, and then sum up the core of the international fishery issue, that is international cooperation with a prerequisite of chasing national interest(chapter 6). / The international conservation of highly migratory species has set out. States are willing to transfer more competence to specialized institution and undertake international cooperation under a certain regulatory framework. This situation deserves much applause. We could look forward that international society would pay more attention to environmental ecological issues to carrying out the idea of sustainable development and management of ocean resource.