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Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第13793号 / 工博第2897号 / 新制||工||1428(附属図書館) / 26009 / UT51-2008-C709 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 今中 忠行, 教授 青山 安宏, 教授 濵地 格 / 学位規則第4条第1項該当

This diploma thesis deals with dynamic of accumulation of non-structural carbohydrates and activity of Rubisco enzyme at elevated concentration of CO2 on beech (Fagus sylvatica L.). Three years old seedlings of beech were cultivated in minisphere with ambient (385 µmol•mol-1, variant A), and with elevated concentration CO2 (700 µmol•mol-1, variant E) for four months. In every variant the first half of plants was fertilized by nitrogen (variant N+) and the second half was control (variant N-). Plants used for experiment were at first adapted for darkness for 12 hours. Subsequently tested leaves were cut off, leafstalk including short segment of branch (approximately 1 cm) was inserted into 0.7 M solution of sucrose (variant S) or water (variant V) and exposed to radiation 200 mol•m-2•s-1 for 0, 30, 60, 120, 180 minutes. Then leaf area and fresh mass of leaf blade were established, samples were fixed in liquid nitrogen and stored in deep freezer to analysis in –70 °C. Rubisco content was determined by SDS-PAGE method, Rubisco activity spectrofotometrically and content of non-structural carbohydrates by anthrone method and HPLC method. Rubisco content was significantly lower in the N- variant than in N+ variant. Rubisco content was also significantly lower in E than in A variant, which is an evidence of down-regulation. Rubisco activity is moderately stimulated at E variant with time, but differences between variant A and E are not statistically significant. Influence of sucrose feeding to Rubisco activity was not proved. Significant differences were detected by anthrone method in non-structural carbohydrates content between variants S and V, but not between variants A and E. Statistically significant increase of sucrose content with time was detected by HPLC method at variant AS, but not at variant ES.

Characterization of Rubisco plays a crucial role when it comes to the development and understanding of carbon sequestration in plants. This project took place at BMC in Uppsala, in the Gunn lab, and aimed to structure three Rubisco structures and analyze these with regard to the assembly pathway of the biogenesis of Rubisco but also how fast the reaction of binding of atmospheric carbon dioxide takes place with regard to different isoforms of the small subunit. The structural regulations led to the conclusion that an additional step in the assembly pathway would be added when one side of Rubisco had the chaperone BSD2 bound while the other side of Rubisco had the small subunit bound.The different subunits are believed to effect the structure of the LSu. The result also indicate that when the SSu are binding to the LSu octomer the interactions between the BSD2 and the LSu changes. This indicats that the SSu could indirectly facilitate the binding of the SSu on the other side by affecting the interactions of the LSu and the BSD2. Therefore the cooperative binding of the different subunits would be interesting to further evaluate. The NtL8B4(S-T1)4 , which is the first model for this structure to be determined, and therefore extended the assembly pathway for the biogenesis of higher plants, had the CABP bound, indicating that this intermediate structure could be analytically competent. This hypothesis is only based on the analyses of the structural determination, therefore further studies are needed to determine whether this is legitimate. Teknisk-naturvetenskapliga fakulteten, Uppsala universitet. Utgivni

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Ribulose-1,5-Bifsfosfato (RuBP) carboxilase/oxigenase (RuBisCO) é a enzima chave para a fixação do carbono atmosférico e para a produtividade das plantas. Não há, até o momento, uma metodologia estabelecida para otimizar o processo de fixação do CO2 nas diferentes espécies de plantas. Entretanto, a disponibilidade de um protocolo de transformação genética de cloroplasto de tabaco permite tentativas de manipulação da enzima RuBisCO visando aumento da eficiência fotossintética. Nas plantas, esta proteína é formada por 8 subunidades menores codificadas pelo gene rbcS localizado no genoma nuclear e por 8 subunidades maiores codificadas pelo gene rbcL localizado no genoma de cloroplastos. Neste trabalho, dois genes rbcL-sintéticos, um com a substituição da alanina (A) 378 por uma valina (V) (A378V) e outro sem a substituição foram utilizados para a construção dos vetores pTT629, pTT630, pTT632 e pTT633. Estes vetores foram usados para transformar o cloroplasto de folhas de tabaco, pelo método de biolística. Um total de 35 plantas transplastômicas se desenvolveram sob seleção dos antibióticos espectinomicina (500 mg/L) e estreptomicina (500 mg/L) e a análise molecular dos sítios de restrição AccI, EcoRI, NdeI e NsiI, de fragmentos amplificados da sequência codante atpB::rbcL:: aadA:: accD demonstrou a integração dos genes rbcL-sintéticos em 11 linhagens transplastômicas. Sementes F1 destas plantas demonstraram ser homoplásmicas pela germinação na presença do antibiótico espectinomicina (500 mg/L). Análises fisiológicas das taxas de fotossíntese (A), condutância estomática (gs) e de transpiração (E) das plantas transplastômicas (A378V) mantidas em casa-de-vegetação produziram valores maiores e significativos, quando comparados com as plantas sem a mutação e controle não transgênicos. O aumento da taxa de fotossíntese das linhagens transplastômicas indicam a possibilidade de aumento da atividade catalítica da RuBisCO. A compreensão da interação fotossintética com a atividade fotorrespiratória poderá permitir explorar e estender possíveis benefícios, como o aumento da produtividade em cultivares de interesse agronômico. / Ribulose-1,5-Bifsfosfato (RuBP) carboxylase/ oxygenase (RuBisCO) is the key enzyme for the fixation of atmospheric carbon and productivity of plants. At moment, no single solution to optimize the CO2 fixing process by the different species of plants. The availability of a few efficient chloroplast transformation protocols for all cultivars also directs attempts to manipulate the larger and small subunit of RuBisCO. In plants, this protein consists of coding form eight smaller subunits encoding the rbcS gene and 8 larger subunits of the rbcL gene respectively located in the nucleus and chloroplasts. Using two rbcL-synthetic genes, with an alanine (Ala) 378 substituting a valine (Val) (A378V) and another one without the replacement were used in the construction of pTT629, pTT630, pTT632 and pTT633 vectors, which were used in the method of biolistic to driving these transgenes into the chloroplast genome of tobacco. A total of 35 transplastomic plants were grown under selection of antibiotics spectinomycin (500mg/ L) and streptomycin (500mg/ L) and the molecular analysis using restriction sites AccI, EcoRI, NdeI and NsiI from the amplified fragments of atpB::rbcL:: aadA:: accD sequence displayed the rbcL-synthetic genes integrated into the plastome of the 11 transplastomic lines. The F1 seeds of these plants were shown to be homoplasmic from germinating in the presence of the antibiotic spectinomycin (500mg / L). The physiology analyzes of photosynthesis (A), stomatal conductance (gs) and transpiration (E) rates of these transplastomic lines (A378V) plants kept in green-house produced the highest and significant values when when compared to the control plants without the mutation and non-transgenic control. The increase of the photosynthesis rate form transplastomic lines indicates the possibility of increasing the catalytic activity of RuBisCO. The understanding of the photosynthetic interaction with photorespiration activity may allow explore more the potential benefits, such as increased productivity in crops of agronomic interest.

This thesis contains quantum chemical models and force field calculations for the RuBisCO isotope effect, the spectral characteristics of the blue-light sensor BLUF and the light harvesting complex II. The work focuses on the influence of the environment on the corresponding systems. For RuBisCO, it was found that the isotopic effect is almost unaffected by the environment. In case of the BLUF domain, an amino acid was found to be important for the UV/vis spectrum, but unaccounted for in experiments so far (Ser41). The residue was shown to be highly mobile and with a systematic influence on the spectral shift of the BLUF domain chromophore (flavin). Finally, for LHCII it was found that small changes in the geometry of a Chlorophyll b/Violaxanthin chromophore pair can have strong influences regarding the light harvesting mechanism. Especially here it was seen that the proper description of the environment can be critical. In conclusion, the environment was observed to be of often unexpected importance for the molecular properties, and it seems not possible to give a reliable estimate on the changes created by the presence of the environment. / Diese Arbeit beinhaltet quantenchemische und molekularmechanische Modelle zum Isotopeneffekt des Enzyms RuBisCO, der spektralen Charakterisierung des Blaulicht-Rezeptors BLUF und dem Lichtsammelkomplex II (LHCII). Es wurden vor allem die Einflüsse der Umgebung auf die entsprechenden Systeme untersucht. Für RuBisCO wurde gefunden, dass der Isotopeneffekt nur marginal von der Umgebung abhängt. Im Falle der BLUF Domäne wurde eine Aminosäure charakterisiert (Ser41), die bis dato experimentell noch nicht beschrieben war. Es wurde festgestellt, dass Ser41 hochmobil ist und einen systematischen Einfluss auf die spektrale Verschiebung des BLUF Chromophors (Flavin) hat. Schließlich wurde bei LHCII festgestellt, dass kleine Veränderungen in der Geometrie eines Chlorophyll b/Violaxanthin Chromophorenpaares bereits massive Einflüsse auf den Mechanismus des Lichtsammelprozesses haben können. Insbesondere hier zeigt sich, wie kritisch die genaue Beschreibung der Umgebung ist. Zusammenfassend wurde beobachtet, dass sich die Umgebung in oft unerwarteter Weise auf die molekularen Eigenschaften auswirken kann und es daher nicht möglich zu sein scheint, die entsprechenden Effekte vorher abzuschätzen.