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.

Current crop and climate modeling studies predict temperature extremes that may add future challenges to global food and agriculture production systems due to yield decreases in our staple cereal crops. Although there have been some temperature stress adaptation traits and a few associated genes discovered in plants for enhanced thermotolerance, very little is known about these traits in our major cereal crops, particularly in maize. Furthermore, the limited availability of appropriate selection environments and accurate phenotyping, including functional traits for selection, have been major hurdles to overcome in making meaningful gains towards improved thermotolerance in breeding programs. Previous reports have established that dynamic changes in leaf membrane lipids occur when exposed to temperature stress and many have tried to identify specific lipid classes, individual species, or levels of unsaturation as indicators of tolerance or susceptibility. In this study, several types of cereals, with special emphasis on maize, are studied with the goal to expand the understanding of the leaf lipid membrane characteristics and responses when exposed to temperature stress and to find evidence of heritable lipid biomarker(s) that could be used in breeding for enhanced thermotolerance. Leaf lipids for maize inbred lines and twelve hybrids exhibiting differential tolerance to high temperature stress were analyzed after growing in a controlled environment at optimal and high temperature stress conditions. It was hypothesized that the newly introduced ratios of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) containing acyl chains with total 36 carbons and 6 double bonds (36:6) compared to those with 36 carbons and 5 double bonds (36:5), here termed the MGDG and DGDG “Unsaturation Ratios” can be used to differentiate the changes in the plastidic lipid membrane unsaturation levels and to aid in identifying heat tolerant genotypes. An analysis of the MGDG and DGDG Unsaturation Ratios was performed on twenty-five diverse parents of the nested-association mapping (NAM) population, Mo17, and one hundred and ninety-one B73 x B97 recombinant inbred lines (RILs) grown in field conditions. The selected lipid phenotypes were found to be as diverse as the inbred lines in which they were measured and showed a large differential between the temperate inbred lines B73 and B97. Allelic variation controlling the differences in MGDG and DGDG Unsaturation Ratios was identified in the B73 x B97 RIL subpopulation through linkage mapping analysis. Finally, an analysis of the MGDG and DGDG Unsaturation Ratios was performed across eight of the world’s most important cereal crops. The results of these studies provide preliminary evidence that the MGDG and DGDG Unsaturation Ratios may be beneficial lipid biomarkers that can be used to screen germplasm in breeding programs for improved thermotolerance for our most important cereal crops with the potential to differentiate tolerance in germplasm even without the presence of the ideal selection environment.

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.

Master of Science / Department of Plant Pathology / William W. Bockus / The effects of tan spot on lipid profiles in wheat leaves were quantified by mass spectrometry. Inoculation with Pyrenophora tritici-repentis significantly reduced the amount of many lipids, including the major lipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), in leaves over time. These two lipids accounted for 89% of the mass spectral signal of detected lipids in wheat leaves. Reductions in amounts of lipids were at much higher rates over time for susceptible cultivars compared with resistant cultivars. Furthermore, data show that cultivars resistant to tan spot have different lipid profiles when compared with susceptible cultivars. Resistant cultivars had more MGDG and DGDG than susceptible ones, even in non-inoculated leaves. Using linear models that were fit to data, non-inoculated cultivars with a rating of 1 (highly resistant to tan spot) were calculated to have 66.1% more MGDG and 52.7% more DGDG signal than cultivars with a rating of 9 (highly susceptible). These latter findings are indirect evidence that the amounts of some lipids in wheat leaves may be determining factors in the resistance response of cultivars to tan spot.

Dans de nombreux sols, le phosphate est un élément limitant pour la croissance des plantes. Au niveau cellulaire, la carence de phosphate induit une diminution de la teneur en phospholipides, permettant la mobilisation du phosphate contenu dans ces molécules. Cette baisse est compensée par une augmentation de la teneur en glycolipides plastidiaux non phosphorés tels que le digalactosyldiacylglycérol (DGDG). Nous avons montré qu'au cours de la carence de phosphate, une partie des phospholipides est reconvertie en phosphatidylcholine (PC), produisant, au temps court de carence, une accumulation transitoire de PC dans les cellules. La PC est ensuite hydrolysée en diacylglycérol (DAG) qui s'accumule en carence de phosphate et nourrit la synthèse du DGDG. Nos résultats suggèrent un transfert direct du DAG à partir des membranes non plastidiales vers l'enveloppe des plastes, lieu de synthèse du DGDG. Le DGDG est ensuite exporté dans des membranes extraplastidiales. Nous avons mis en évidence la présence de DGDG dans les mitochondries et son transfert des plastes vers les mitochondries à partir de contacts entre des domaines spécialisés de l'enveloppe des plastes et des mitochondries. Enfin, pour identifier des protéines impliquées dans ces mécanismes de remaniement des lipides, nous avons collaboré à une analyse transcriptomique du génome d'Arabidopsis thaliana en carence de phosphate. Nous avons notamment sélectionné une phospholipase D, PLDzéta2, qui semble impliquée dans le contrôle de la teneur intracellulaire en phosphate inorganique et dans l'hydrolyse de la PC pour l'approvisionnement en DAG de la synthèse des galactolipides.