FRANKLIN, Maria Lucia Torres. Estudo do metabolismo dos lipídeos de membranas do cloroplasto e dos genes associados em Vigna unguiculata (L.) Walp. em condição de déficit hídrico e reidratação subseqüente. 2008. 153 f. Tese (Doutorado em bioquímica)- Universidade Federal do Ceará, Fortaleza-CE, 2008. / Submitted by Elineudson Ribeiro (elineudsonr@gmail.com) on 2016-07-20T18:09:28Z No. of bitstreams: 1 2008_tese_mltfranklin.pdf: 2107179 bytes, checksum: 0fd5898d4f6562339271427743469225 (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-07-26T20:07:18Z (GMT) No. of bitstreams: 1 2008_tese_mltfranklin.pdf: 2107179 bytes, checksum: 0fd5898d4f6562339271427743469225 (MD5) / Made available in DSpace on 2016-07-26T20:07:18Z (GMT). No. of bitstreams: 1 2008_tese_mltfranklin.pdf: 2107179 bytes, checksum: 0fd5898d4f6562339271427743469225 (MD5) Previous issue date: 2008 / Membranes are main targets of degradation by reactive oxygen species and hydrolytic activities induced by drought. Chloroplasts lipid biosynthesis, especially galactolipids monogalactosyl-diacylglycerol (MGDG) and digalactosyl-diacylglycerol (DGDG) are important for plant tolerance to water deficit and for recovery after rehydration. In this thesis, we studied the metabolism of the chloroplast membrane lipids, MGDG, DGDG, sulphoquinovosyl-diacylglycerol (SQDG), phosphatidyl-glycerol (PG) under drought and during recovery from drought. Aiming this, we measured leaf lipids content, followed 14C-acétate incorporation and expression of genes coding for chloroplast membrane lipid synthases (MGD1, MGD2, DGD1, DGD2, SQD2 and PGP1) during drought and recovery. In order to better understand the relationship between drought tolerance and lipid metabolism, two cultivars of Vigna unguiculata L. Walp, one drought tolerant (cv. EPACE) the other drought susceptible (cv. 1183) were compared. The cDNA complete sequences for VuMGD1, VuMGD2, VuDGD1, VuDGD2, VuSQD2 and VuPGP1 were obtained from screening of a V.unguiculata cDNA library. The results showed that under water stress conditions, the tolerant cultivar, besides its ability to preserve its lipids pool despites drought, is able to strongly stimulate the DGDG biosynthesis, increasing the DGDG:MGDG ratio in its membranes. We suggest that DGDG accumulated under drought condition, when phosphate is deficient, is exported for extrachloroplastic membranes, and thus contributes to plant drought tolerance. Effects of loss of water on cell membranes have direct consequences on plant capacity to recover from stress. 48 hours after rewatering, the susceptible cv. 1183 was not able to fully recover from a moderate stress in terms of leaf galactolipid content and acetate incorporation into MGDG. In EPACE-1, MGDG leaf content remained unchanged after rehydration and DGDG remained higher than in the control plants. In conclusion, our results highlight the importance of membrane lipids in plant adaptation to water deficit and in their capacity to recover from stress. Of particular importance is the balance between lipid classes with various physico-chemical properties (SQDG versus PG, DGDG versus MGDG), since they most likely have a profound influence on membrane structure and function. / As membranas biológicas são alvos preferenciais dos efeitos deletérios do estresse hídrico, induzidos por ação de enzimas hidrolíticas e espécies reativas do oxigênio (ERO), ambas estimuladas durante o estresse. A biossíntese dos lipídeos dos cloroplastos pode ser importante para a tolerância ao estresse hídrico e para a recuperação após reidratação. Nesse trabalho nos estudamos o metabolismo dos cloroplastos, monogalactosil-diacilglicerol (MGDG), digalactosil-diacilglicerol (DGDG), sulfoquinovosil-diacilglicerol (SQDG), phosphatidil-glicerol (PG), no âmbito do déficit hídrico e da reidratação após o fim do estresse. Com este intuito, nos medimos o teor dos lipídeos da folhas, acompanhamos a incorporação do precursor 14C-acetato nos lipídeos e analisamos a expressão dos genes codificadores das enzimas de síntese chave dos lipídeos (MGD1, MGD2, DGD1, DGD2, SQD2 e PGP1) durante o estresse hídrico e após a reidratação. Visando de uma melhor compreensão da relação entre o metabolismo destes lipídeos e a tolerância a seca, nos trabalhamos com duas cultivares de Vigna unguiculata L. Walp, uma tolerante (cv. EPACE) e outra sensível (cv. 1183) a seca. Por meio de varredura diferencial de um biblioteca de cDNA de V.unguiculata, foram obtidas as seqüências completas dos cDNA dos genes VuMGD1, VuMGD2, VuDGD1, VuDGD2, VuSQD2 e VuPGP1. Os resultados mostram que em condições de estresse hídrico a cultivar tolerante, além de preservar seu teor de lipídeos durante a seca, é igualmente capaz de estimular a biossíntese do DGDG aumentando significativamente a relação DGDG:MGDG de suas membranas. Nós sugerimos que o DGDG acumulado em condição de seca é transportado para as membranas externas ao cloroplasto e que isso contribui para a tolerância à seca. Os efeitos da desidratação celular sobre as membranas têm conseqüências diretas sobre a capacidade das plantas a se recuperarem após reidratação. 48 horas após a rega, a cv. sensível 1183 não é capaz de se recuperar em termos de teor de galactolipídeos e incorporação do precursor. Na cv. tolerante, no entanto, o teor de DGDG permanece elevado, mesmo após a reidratação. Em conclusão, nossos resultados sugerem a importância dos lipídeos membranares na tolerância/sensibilidade das plantas ao déficit hídrico, em particular o balanço entre as classes lipídicas de propriedades físico-químicas diferentes (SQDG versus PG e DGDG versus MGDG) que poderiam afetar a estrutura e o funcionamento das membranas.

Les membranes cellulaires sont des cibles préférentielles de la dégradation induite par les espèces réactives de l’oxygène produites durant la sécheresse et par la stimulation d’activités hydrolytiques. La biosynthèse des lipides des chloroplastes peut être importante pour la tolérance à la sécheresse ainsi que pour la reprise après réhydratation. Dans ce travail nous avons étudié le métabolisme des lipides des membranes chloroplastiques, monogalactosyldiacylglycerol (MGDG), digalactosyl-diacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), phosphatidyl-glycerol (PG), dans le cadre de la sécheresse et de la reprise après la fin de la contrainte hydrique. Dans ce but, nous avons mesuré la teneur des lipides des feuilles, suivi l’incorporation du précurseur 14C-acétate dans les lipides et analysé l’expression des gènes codant les enzymes de biosynthèse des lipides (MGD1, MGD2, DGD1, DGD2, SQD2 et PGP1) durant le stress hydrique et après réhydratation. Afin de mieux comprendre le rapport entre le métabolisme de ces lipides et la tolérance à la sécheresse, nous avons travaillé sur deux cultivars de Vigna unguiculata L. Walp, un tolérant (cv. EPACE) et l’autre sensible (cv. 1183) à la sécheresse. Les séquences complètes des ADNc des gènes VuMGD1, VuMGD2, VuDGD1, VuDGD2, VuSQD2 et VuPGP1 ont été obtenues par le criblage d’une banque d’ADNc de V.unguiculata. Les résultats montrent qu’en condition de stress hydrique le cultivar tolérant, en plus de préserver la teneur en lipides, est capable de stimuler la biosynthèse du DGDG augmentant significativement le rapport DGDG:MGDG de ces membranes. Nous suggérons que le DGDG accumulé en sécheresse est exporté vers les membranes extrachloroplastiques et que cela contribue à la tolérance à la contrainte hydrique. Les effets de la perte d’eau cellulaire sur les membranes ont des conséquences directes sur la capacité des plantes à reprendre après réhydratation. 48 heures après réarrosage, le cv. sensible 1183 n’est pas capable de récupérer en termes de teneurs en galactolipides et incorporation de précurseur. Chez le cv. tolérant, par contre, la teneur en DGDG demeure élevé, même après réhydratation. En conclusion, nos résultats suggèrent l’importance des lipides membranaires dans la tolérance/sensibilité des plantes au déficit hydrique, en particulier la balance entre des classes lipidiques de propriétés physico-chimiques différentes (SQDG versus PG et DGDG versus MGDG) qui pourraient affecter la structure et le fonctionnement des membranes / Membranes are main targets of degradation by reactive oxygen species and hydrolytic activities induced by drought. Chloroplasts lipid biosynthesis, especially galactolipids monogalactosyl-diacylglycerol (MGDG) and digalactosyl-diacylglycerol (DGDG) are important for plant tolerance to water deficit and for recovery after rehydration. In this thesis, we studied the metabolism of the chloroplast membrane lipids, MGDG, DGDG, sulphoquinovosyl-diacylglycerol (SQDG), phosphatidyl-glycerol (PG) under drought and during recovery from drought. Aiming this, we measured leaf lipids content, followed 14Cacétate incorporation and expression of genes coding for chloroplast membrane lipid synthases (MGD1, MGD2, DGD1, DGD2, SQD2 and PGP1) during drought and recovery. In order to better understand the relationship between drought tolerance and lipid metabolism, two cultivars of Vigna unguiculata L. Walp, one drought tolerant (cv. EPACE) the other drought susceptible (cv. 1183) were compared. The cDNA complete sequences for VuMGD1, VuMGD2, VuDGD1, VuDGD2, VuSQD2 and VuPGP1 were obtained from screening of a V.unguiculata cDNA library. The results showed that under water stress conditions, the tolerant cultivar, besides its ability to preserve its lipids pool despites drought, is able to strongly stimulate the DGDG biosynthesis, increasing the DGDG:MGDG ratio in its membranes. We suggest that DGDG accumulated under drought condition, when phosphate is deficient, is exported for extrachloroplastic membranes, and thus contributes to plant drought tolerance. Effects of loss of water on cell membranes have direct consequences on plant capacity to recover from stress. 48 hours after rewatering, the susceptible cv. 1183 was not able to fully recover from a moderate stress in terms of leaf galactolipid content and acetate incorporation into MGDG. In EPACE-1, MGDG leaf content remained unchanged after rehydration and DGDG remained higher than in the control plants. In conclusion, our results highlight the importance of membrane lipids in plant adaptation to water deficit and in their capacity to recover from stress. Of particular importance is the balance between lipid classes with various physico-chemical properties (SQDG versus PG, DGDG versus MGDG), since they most likely have a profound influence on membrane structure and function

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / As membranas biolÃgicas sÃo alvos preferenciais dos efeitos deletÃrios do estresse hÃdrico, induzidos por aÃÃo de enzimas hidrolÃticas e espÃcies reativas do oxigÃnio (ERO), ambas estimuladas durante o estresse. A biossÃntese dos lipÃdeos dos cloroplastos pode ser importante para a tolerÃncia ao estresse hÃdrico e para a recuperaÃÃo apÃs reidrataÃÃo. Nesse trabalho nos estudamos o metabolismo dos cloroplastos, monogalactosil-diacilglicerol (MGDG), digalactosil-diacilglicerol (DGDG), sulfoquinovosil-diacilglicerol (SQDG), phosphatidil-glicerol (PG), no Ãmbito do dÃficit hÃdrico e da reidrataÃÃo apÃs o fim do estresse. Com este intuito, nos medimos o teor dos lipÃdeos da folhas, acompanhamos a incorporaÃÃo do precursor 14C-acetato nos lipÃdeos e analisamos a expressÃo dos genes codificadores das enzimas de sÃntese chave dos lipÃdeos (MGD1, MGD2, DGD1, DGD2, SQD2 e PGP1) durante o estresse hÃdrico e apÃs a reidrataÃÃo. Visando de uma melhor compreensÃo da relaÃÃo entre o metabolismo destes lipÃdeos e a tolerÃncia a seca, nos trabalhamos com duas cultivares de Vigna unguiculata L. Walp, uma tolerante (cv. EPACE) e outra sensÃvel (cv. 1183) a seca. Por meio de varredura diferencial de um biblioteca de cDNA de V.unguiculata, foram obtidas as seqÃÃncias completas dos cDNA dos genes VuMGD1, VuMGD2, VuDGD1, VuDGD2, VuSQD2 e VuPGP1. Os resultados mostram que em condiÃÃes de estresse hÃdrico a cultivar tolerante, alÃm de preservar seu teor de lipÃdeos durante a seca, à igualmente capaz de estimular a biossÃntese do DGDG aumentando significativamente a relaÃÃo DGDG:MGDG de suas membranas. NÃs sugerimos que o DGDG acumulado em condiÃÃo de seca à transportado para as membranas externas ao cloroplasto e que isso contribui para a tolerÃncia à seca. Os efeitos da desidrataÃÃo celular sobre as membranas tÃm conseqÃÃncias diretas sobre a capacidade das plantas a se recuperarem apÃs reidrataÃÃo. 48 horas apÃs a rega, a cv. sensÃvel 1183 nÃo à capaz de se recuperar em termos de teor de galactolipÃdeos e incorporaÃÃo do precursor. Na cv. tolerante, no entanto, o teor de DGDG permanece elevado, mesmo apÃs a reidrataÃÃo. Em conclusÃo, nossos resultados sugerem a importÃncia dos lipÃdeos membranares na tolerÃncia/sensibilidade das plantas ao dÃficit hÃdrico, em particular o balanÃo entre as classes lipÃdicas de propriedades fÃsico-quÃmicas diferentes (SQDG versus PG e DGDG versus MGDG) que poderiam afetar a estrutura e o funcionamento das membranas. / Membranes are main targets of degradation by reactive oxygen species and hydrolytic activities induced by drought. Chloroplasts lipid biosynthesis, especially galactolipids monogalactosyl-diacylglycerol (MGDG) and digalactosyl-diacylglycerol (DGDG) are important for plant tolerance to water deficit and for recovery after rehydration. In this thesis, we studied the metabolism of the chloroplast membrane lipids, MGDG, DGDG, sulphoquinovosyl-diacylglycerol (SQDG), phosphatidyl-glycerol (PG) under drought and during recovery from drought. Aiming this, we measured leaf lipids content, followed 14C-acÃtate incorporation and expression of genes coding for chloroplast membrane lipid synthases (MGD1, MGD2, DGD1, DGD2, SQD2 and PGP1) during drought and recovery. In order to better understand the relationship between drought tolerance and lipid metabolism, two cultivars of Vigna unguiculata L. Walp, one drought tolerant (cv. EPACE) the other drought susceptible (cv. 1183) were compared. The cDNA complete sequences for VuMGD1, VuMGD2, VuDGD1, VuDGD2, VuSQD2 and VuPGP1 were obtained from screening of a V.unguiculata cDNA library. The results showed that under water stress conditions, the tolerant cultivar, besides its ability to preserve its lipids pool despites drought, is able to strongly stimulate the DGDG biosynthesis, increasing the DGDG:MGDG ratio in its membranes. We suggest that DGDG accumulated under drought condition, when phosphate is deficient, is exported for extrachloroplastic membranes, and thus contributes to plant drought tolerance. Effects of loss of water on cell membranes have direct consequences on plant capacity to recover from stress. 48 hours after rewatering, the susceptible cv. 1183 was not able to fully recover from a moderate stress in terms of leaf galactolipid content and acetate incorporation into MGDG. In EPACE-1, MGDG leaf content remained unchanged after rehydration and DGDG remained higher than in the control plants. In conclusion, our results highlight the importance of membrane lipids in plant adaptation to water deficit and in their capacity to recover from stress. Of particular importance is the balance between lipid classes with various physico-chemical properties (SQDG versus PG, DGDG versus MGDG), since they most likely have a profound influence on membrane structure and function.