Made available in DSpace on 2015-03-26T13:36:40Z (GMT). No. of bitstreams: 1 texto completo.pdf: 450533 bytes, checksum: d14de51f177a16c047cb046fafb1e533 (MD5) Previous issue date: 2009-02-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Mutations in GUN (Genome UNcoupled) genes have helped to understand the pathways of the chloroplast signal transduction that control the expression of nuclear photosynthetic genes. In this context, the gun4 genotype has been characterized in biochemical and molecular aspects. However, little is known whether this mutant produces a phenotype physiological aspects. In order to complement the phenotypic characterization of gun4 genotype, the aim of this study was evaluate the photosynthetic responses of gun4 plants of Arabidopsis thaliana under different levels of light and under light stress. The gun4 plants, in the absence of light stress showed reduced levels of chlorophyll a and b, minimal (Fo) and maximal (Fm) fluorescence in dark-adapted leaves, and quantum yield of unregulated energy dissipation of photosystem II (ΦNO) in 20%, 26%, 18% e 32%, respectively. Also, the effective quantum yield of the FSII (ΦFSII) and photochemical quenching (qL) were 11% and 20% higher, respectively. Higher values of ETR (electrons transport rate), ΦFSII and qL were also found in gun4 under different light intensities, showing that gun4 can maximize the transfer of light energy absorbed in the light harvest complex of FSII for the photosynthetic process. The rate of net carbon assimilation (A) and stomatal conductance (gs) were 43% and 61% lower in gun4 respectively, despite no difference in the flow of absorption of 14CO2 was observed, suggesting that the lower magnitude of photosynthesis is due to resistance to influx of CO2. After 14 hours of light stress, there was no difference in A, and the values of the electron transport rate (ETR) and ΦFSII were reduced by 35% and 50% respectively, both in gun4 and wild type (WT) plants. However, the quantum yield potential of FSII (Fv/Fm) decreased significantly only in WT, suggesting that gun4 may have a greater ability to minimize photoinhibitory effects. These results provide additional evidences for the higher non-photochemical quenching (qN) and quantum yield of regulated energy dissipation of the gun4 FSII (ΦNPQ). Together, the photochemical phenotypes of gun4 may explain the increase (48%) in the photochemical global network connectance after 28 hours of stress, demonstrating the greater ability of this genotype to adjust its photosynthetic apparatus in response to light stress. Finally, the results found in this study demonstrated that gun4 transfers and dissipates more efficiently the excess of light energy absorbed under light stress condition, despite a strong reduction in chlorophyll content. The results suggest the existence of a mechanism for the adjustment of the photosystem in order to offset the reduction of light absorption, indicating the importance of this mutant to explore the mechanisms that control the plasticity of the photochemical protein complex. / Mutações em genes denominados GUN (Genome UNcoupled) têm auxiliado na compreensão das rotas de transdução de sinais do cloroplasto que controlam a expressão de genes fotossintéticos nucleares. Nesse sentido, o genótipo gun4 tem sido caracterizado em aspectos bioquímicos e moleculares, no entanto pouco se sabe sobre aspectos fisiológicos desse mutante. Diante disso, de forma a complementar essa caracterização fenotípica, o objetivo desse trabalho foi avaliar as respostas fotossintéticas do mutante gun4 de Arabidopsis thaliana sob diferentes níveis de luz. Plantas gun4, na ausência de estresse luminoso, apresentaram níveis de clorofila a e b, fluorescências mínima (Fo) e máxima (Fm) de folhas adaptadas ao escuro e rendimento quântico de dissipação não regulada de energia do fotossistema II (ΦNO) reduzidos em 20%, 26%, 18% e 32%, respectivamente, enquanto que o rendimento quântico efetivo do FSII (ΦFSII) e o coeficiente de extinção fotoquímico (qL) foram 11% e 20% maiores, respectivamente. Valores maiores de ETR (taxa de transporte de elétrons), ΦFSII e qL também foram observados em gun4 sob diferentes intensidades luminosas, demonstrando que este fenótipo transfere de forma mais eficiente a energia luminosa absorvida no complexo coletor de luz do FSII para o processo fotossintético. A taxa de assimilação líquida de carbono (A) e a condutância estomática (gs) foram 43% e 61% menores em gun4, respectivamente. Porém, não houve diferença no fluxo de absorção de 14CO2, sugerindo que a menor magnitude de A seja devido a uma restrição difusional ao influxo de CO2. Após 14 h de estresse, não houve diferença em A e os valores da taxa de transporte de elétrons (ETR) e ΦFSII foram reduzidos em 35% e 50%, respectivamente, tanto nas plantas gun4 como no tipo selvagem (WT). Entretanto, o rendimento quântico potencial do FSII (Fv/Fm) decresceu significativamente apenas em WT, sugerindo que gun4 possa minimizar, mais eficientemente, efeitos fotoinibitórios. Isto se evidencia, adicionalmente, nos maiores valores de coeficiente de extinção não fotoquímico (qN) e rendimento quântico de dissipação regulada de energia do FSII (ΦNPQ). Em conjunto, esses fenótipos fotoquímicos de gun4 podem explicar o maior aumento (48%) na conectância global da rede fotoquímica após 28 h de estresse, demonstrando a maior capacidade deste genótipo em ajustar seu aparato fotossintético em resposta ao estresse luminoso. Por fim, os resultados encontrados neste trabalho demonstraram a alta capacidade do genótipo gun4 em dissipar o excesso de energia luminosa absorvida sob estresse e, apesar da forte redução no teor de clorofila, transferir esta mais eficientemente. Isso sugere a existência de um mecanismo de adaptação dos fotossistemas de forma a compensar a redução na absorção de luz, indicando um valor importante deste mutante para explorar os mecanismos que controlam a plasticidade dos complexos protéicos fotoquímicos.

Im ersten Teil dieser Studie habe ich gezeigt, dass die Chaperonaktivität von cpSRP43 für die Stabilität der drei essenziellen TBS-Proteine Glutamyl-tRNA-Reduktase (GluTR), der H-Untereinheit der Magnesium-Chelatase (CHLH) und von Genomes Uncoupled 4 (GUN4 ) wichtig ist. cpSRP43 schützt diese TBS-Proteine effizient vor hitzeinduzierter Aggregatbildung und verbessert ihre Thermostabilität während eines Hitzeschocks. Während die substratbindende Domäne (SBD) von cpSRP43 für die Interaktion mit LHCPs ausreicht, erfordert die Stabilisierung der TBS-Proteine die zusätzliche cpSRP43-Chromodomäne 2 (CD2). Es wurde überraschend gefunden, dass cpSRP54 die Chaperonaktivität von cpSRP43 für LHCPs aktiviert, während es sie für TBS-Proteine vermindern kann. Aber erhöhte Temperatur kann die Bindung von cpSRP43 mit cpSRP54 lösen, aber seine Wechselwirkung mit GluTR, CHLH und GUN4 verstärken, was zu einem verstärkten Schutz von cpSRP43 gegenüber diesen Proteinen unter Hitzeschockbedingungen führt. Im zweiten Teil dieser Studie wurde festgestellt, dass PORB (eines der drei POR Isoformen) ein weiteres Ziel von cpSRP43 ist. Zusammenfassend haben meine Studien einen möglichen Mechanismus von PORB durch konzertierte Aktionen von cpSRP43 und cpSRP54 aufgezeigt. (1) cpSRP43 wirkt als molekulares Chaperon, um PORB vor der Aggregation zu schützen, wodurch die Stabilität des PORB bewahrt wird. (2) cpSRP54 kann PORB bei der Anlagerung an die Thylakoidmembran unterstützen, was vermutlich den Abbau von PORB vermeidet und die Stabilität von PORB verbessert oder den Zugang zum katalytischen Substrat ermöglicht. Zusammenfassend trägt diese Arbeit zum erweiterten Wissen über die voneinander abhängige Chaperonfunktion der beiden Proteine cpSRP43 und cpSRP54 bei der Koordination von Chlorophyllsynthese und LHCP-Biogenese bei. / In the first part of this study, I showed that the chaperone activity of cpSRP43 is essential for the stability of the three critical TBS proteins: glutamyl-tRNA reductase (GluTR), the H subunit of magnesium chelatase (CHLH), and GENOMES UNCOUPLED 4 (GUN4). cpSRP43 efficiently protects these TBS clients from heat-induced aggregation and enhances their thermostability during heat shock. Although the substrate-binding domain (SBD) of cpSRP43 is sufficient for the interaction with LHCPs, the stabilization of TBS clients requires the additional chromodomain 2 (CD2). cpSRP54, which activates the chaperone activity of cpSRP43 on LHCPs, was surprisingly found to antagonize this chaperone activity on TBS proteins. The elevated temperature alleviates the binding of cpSRP43 to cpSRP54 but enhances its interaction with GluTR, CHLH and GUN4, resulting in enhanced protection of cpSRP43 to these proteins under heat shock conditions. My study suggests a working model that the temperature sensitivity of the cpSRP43-cpSRP54 complex enables cpSRP43 to serve as an autonomous chaperone for the thermoprotection of TBS proteins. In the second part of this study, PORB (one of the three POR isoforms) was found to be a new target of cpSRP43. My study revealed a potential mechanism of PORB by concerted actions of cpSRP43 and cpSRP54: (1) cpSRP43 acts as a molecular chaperone to protect PORB from aggregation, thereby preserving the stability of PORB. (2) cpSRP54 assists PORB in attachment to the thylakoid membrane, avoids the degradation of PORB and, thus, improves the stability of PORB or enables access to the catalytic substrate. In summary, this thesis contributes to the extended knowledge about the interdependent chaperone function of cpSRP43 and cpSRP54 in coordination of Chl synthesis and LHCP biogenesis.