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PAIVA, Ana Luiza Sobral. Respostas bioquímicas do feijão-de-corda [Vigna unguiculata L. (Walp.)] ao estresse salino e infecção pelo vírus do mosaico severo do caupi (CPSMV) reveladas pela proteômica quantitativa livre de marcação. 2015. 200 f. Dissertação (Mestrado em Bioquímica) - Universidade Federal do Ceará, Fortaleza-CE, 2015. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-07-08T12:56:09Z No. of bitstreams: 1 2015_dis_alspaiva.pdf: 4225070 bytes, checksum: 0559261a4c594b2649cdb60e4563c1fc (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-08-02T20:16:37Z (GMT) No. of bitstreams: 1 2015_dis_alspaiva.pdf: 4225070 bytes, checksum: 0559261a4c594b2649cdb60e4563c1fc (MD5) / Made available in DSpace on 2016-08-02T20:16:37Z (GMT). No. of bitstreams: 1 2015_dis_alspaiva.pdf: 4225070 bytes, checksum: 0559261a4c594b2649cdb60e4563c1fc (MD5) Previous issue date: 2015 / As sessile organisms, plants are exposed to a plethora of environmental stresses to which they must respond to maintain efficient growth and survival. Therefore, in order to improve our understanding on the complex mechanisms involved in the cowpea response to salt stress and to a compatible interaction with the cowpea severe mosaic virus (CPSMV), we used a label-free quantitative proteomic approach to identify the salt and virus responsive proteins in the leaves of the Pitiuba (CE-31) cultivar. The proteins extracted from the leaves (control and treated) 2 and 6 days post-treatment only with salt (DPS), only infected with CPSMV (DPV) or both of them (DPSV) were analyzed using mass spectrometry. At 2 DPS, 350 proteins with at least two-fold differences in abundance, in comparison with controls, were differentially accumulated in the leaves of the salt-treated (80% up and 20% down-accumulated), 281 at 2DPV (25% up and 75% down-accumulated) and 321 at 2 DPSV (45% up and 55% down-accumulated) plants. At 6 DPS, 350 proteins were differentially accumulated in the leaves of the salt-treated (90% up and 10% down-accumulated), 225 at 6 DPV (80% up and 20% down-accumulated) and 315 at 6 DPSV (94% up and 6% down-accumulated) plants. The qualitative analysis showed biochemical differences when the cowpea plants were challenged concurrently with both stresses. To cope with salinity, cowpea increased the abundance of proteins directly involved with the salt tolerance mechanisms. The results indicated that the CPSMV induce the down-accumulating of several proteins to invade and spread in host at early infection period (2 DPV), but at 6 DPV plant can induce accumulation of diverse proteins related with defense, although these strategies can’t avoid the negatives effects of disease. When exposed simultaneously to salt/CPSMV stresses, a balance in protein accumulation involved in many biological process. This is the first work employing this approach in cowpea and providing evidences of the plant biochemical mechanisms involved in the responses of cowpea to these stresses. / Como organismos sésseis, as plantas são expostas a uma variedade de estresses ambientais aos quais devem responder para sobreviverem e se desenvolverem. A fim de melhorar a nossa compreensão sobre os mecanismos complexos envolvidos na resposta do feijão-de-corda ao estresse salino e na interação compatível com o vírus do mosaico severo do caupi (CPSMV), foi utilizada uma abordagem proteômica quantitativa, livre de marcação, para identificar proteínas, responsivas a essess estresses em folhas de feijão-de-corda, cv. CE-31. As proteínas extraídas a partir de folhas primárias, 2 e 6 dias após o tratamento só com o sal (DPS), somente infectadas (DPV), ou sob ação combinada dos dois (DPSV) foram analisadas, usando espectrometria de massas e comparadas com grupo controle. No 2° DPS, foram identificadas 350 proteínas diferencialmente acumuladas (80% aumentaram em abundância e 20% diminuíram), no 2° DPV 281 (25% aumentaram em abundância e 75% diminuíram) e no 2° DPSV 321 (45% aumentaram em abundância e 55% diminuíram). Já no 6° DPS, foram identificadas 350 proteínas diferencialmente acumuladas (90% mostraram aumento em abundância e 10% diminuição), no 6° DPV 225 (80% aumentaram em abundância e 20% diminuíram) e no 6° DPSV 315 proteínas(94% aumentaram em abundância e 6% diminuíram). Para lidar com a salinidade, o cv. CE-31 aumentou a abundância de proteínas envolvidas diretamente com os mecanismos de tolerância ao sal. Em relação à infecção da planta pelo CPSMV, os resultados obtidos indicaram que o vírus induz redução na abundância de várias proteínas nos tempos iniciais de infecção, provavelmente favorecendo a invasão e propagação na planta, mas, no 6° DPSV, a planta recupera sua capacidade de acionar mecanismos de defesa, embora esses já não sejam mais efetivos para evitar o estabelecimento da doença viral. Durante exposição simultânea da planta ao sal e ao vírus, ocorreu um equilíbrio entre o aumento e diminuição em abundância de proteínas envolvidas em diversos processos metabólicos. Esse trabalho é pioneiro nessa abordagem em feijão-de-corda e fornece evidências dos mecanismos bioquímicos envolvidos nas resposta da planta a esses estresses.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / As sessile organisms, plants are exposed to a plethora of environmental stresses to which they must respond to maintain efficient growth and survival. Therefore, in order to improve our understanding on the complex mechanisms involved in the cowpea response to salt stress and to a compatible interaction with the cowpea severe mosaic virus (CPSMV), we used a label-free quantitative proteomic approach to identify the salt and virus responsive proteins in the leaves of the Pitiuba (CE-31) cultivar. The proteins extracted from the leaves (control and treated) 2 and 6 days post-treatment only with salt (DPS), only infected with CPSMV (DPV) or both of them (DPSV) were analyzed using mass spectrometry. At 2 DPS, 350 proteins with at least two-fold differences in abundance, in comparison with controls, were differentially accumulated in the leaves of the salt-treated (80% up and 20% down-accumulated), 281 at 2DPV (25% up and 75% down-accumulated) and 321 at 2 DPSV (45% up and 55% down-accumulated) plants. At 6 DPS, 350 proteins were differentially accumulated in the leaves of the salt-treated (90% up and 10% down-accumulated), 225 at 6 DPV (80% up and 20% down-accumulated) and 315 at 6 DPSV (94% up and 6% down-accumulated) plants. The qualitative analysis showed biochemical differences when the cowpea plants were challenged concurrently with both stresses. To cope with salinity, cowpea increased the abundance of proteins directly involved with the salt tolerance mechanisms. The results indicated that the CPSMV induce the down-accumulating of several proteins to invade and spread in host at early infection period (2 DPV), but at 6 DPV plant can induce accumulation of diverse proteins related with defense, although these strategies canât avoid the negatives effects of disease. When exposed simultaneously to salt/CPSMV stresses, a balance in protein accumulation involved in many biological process. This is the first work employing this approach in cowpea and providing evidences of the plant biochemical mechanisms involved in the responses of cowpea to these stresses. / Como organismos sÃsseis, as plantas sÃo expostas a uma variedade de estresses ambientais aos quais devem responder para sobreviverem e se desenvolverem. A fim de melhorar a nossa compreensÃo sobre os mecanismos complexos envolvidos na resposta do feijÃo-de-corda ao estresse salino e na interaÃÃo compatÃvel com o vÃrus do mosaico severo do caupi (CPSMV), foi utilizada uma abordagem proteÃmica quantitativa, livre de marcaÃÃo, para identificar proteÃnas, responsivas a essess estresses em folhas de feijÃo-de-corda, cv. CE-31. As proteÃnas extraÃdas a partir de folhas primÃrias, 2 e 6 dias apÃs o tratamento sà com o sal (DPS), somente infectadas (DPV), ou sob aÃÃo combinada dos dois (DPSV) foram analisadas, usando espectrometria de massas e comparadas com grupo controle. No 2 DPS, foram identificadas 350 proteÃnas diferencialmente acumuladas (80% aumentaram em abundÃncia e 20% diminuÃram), no 2 DPV 281 (25% aumentaram em abundÃncia e 75% diminuÃram) e no 2 DPSV 321 (45% aumentaram em abundÃncia e 55% diminuÃram). Jà no 6 DPS, foram identificadas 350 proteÃnas diferencialmente acumuladas (90% mostraram aumento em abundÃncia e 10% diminuiÃÃo), no 6 DPV 225 (80% aumentaram em abundÃncia e 20% diminuÃram) e no 6 DPSV 315 proteÃnas(94% aumentaram em abundÃncia e 6% diminuÃram). Para lidar com a salinidade, o cv. CE-31 aumentou a abundÃncia de proteÃnas envolvidas diretamente com os mecanismos de tolerÃncia ao sal. Em relaÃÃo à infecÃÃo da planta pelo CPSMV, os resultados obtidos indicaram que o vÃrus induz reduÃÃo na abundÃncia de vÃrias proteÃnas nos tempos iniciais de infecÃÃo, provavelmente favorecendo a invasÃo e propagaÃÃo na planta, mas, no 6 DPSV, a planta recupera sua capacidade de acionar mecanismos de defesa, embora esses jà nÃo sejam mais efetivos para evitar o estabelecimento da doenÃa viral. Durante exposiÃÃo simultÃnea da planta ao sal e ao vÃrus, ocorreu um equilÃbrio entre o aumento e diminuiÃÃo em abundÃncia de proteÃnas envolvidas em diversos processos metabÃlicos. Esse trabalho à pioneiro nessa abordagem em feijÃo-de-corda e fornece evidÃncias dos mecanismos bioquÃmicos envolvidos nas resposta da planta a esses estresses.

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Doctor of Philosophy / Department of Agronomy / P.V. Vara Prasad / High temperature (HT) and drought are detrimental to crop productivity, but there is limited variability for these traits among wheat ([italics]Triticum aestivum[end italics] L.) cultivars. Five [italics]Aegilops[end italics] species were screened to identify HT (52 accessions) and drought (31 accessions) tolerant species/accessions and ascertaining traits associated with tolerance. Four synthetic wheats were studied to quantify independent and combined effects of HT and drought. [italics]Aegilops[end italics] species were grown at 25/19°C day/night and 18 h photoperiod. At anthesis, HT was imposed by transferring plants to growth chambers set at 36/30°C, whereas in another experiment, drought was imposed by withholding irrigation. Synthetic wheats were grown at 21/15°C day/night and 18 h photoperiod. At anthesis or 21 d after anthesis, plants were exposed to optimum condition (irrigation + 21/15°C), HT (irrigation + 36/30°C), drought (withhold irrigation + 21/15°C), and combined stress (withhold irrigation + 36/30°C). Stresses were imposed for 16 d. High temperature and drought stress significantly decreased chlorophyll, grain number, individual grain weight, and grain yield of [italics]Aegilops[end italics] species (≥ 25%). Based on a decrease in grain yield, [italics]A. speltoides[end italics] and [italics]A. geniculata[end italics] were most tolerant (~ 61% decline), and [italics]A. longissima[end italics] was highly susceptible to HT stress (84% decline). Similarly, [italics]A. geniculata[end italics] had greater tolerance to drought (48% decline) as compared to other species (≥ 73% decline). Tolerance was associated with higher grains spike [superscript]-1 and/or heavier grains. Within [italics]A. speltoides[end italics], accession TA 2348 was most tolerant to HT with 13.5% yield decline and a heat susceptibility index (HSI) 0.23. Among [italics]A. geniculata[end italics], TA 2899 and TA 1819 were moderately tolerant to HT with an HSI 0.80. TA 10437 of [italics]A. geniculata[end italics] was the most drought tolerant accession with 7% yield decline and drought susceptibility index 0.14. Irrespective of the time of stress, HT, drought, and combined stress decreased both individual grain weight and grain yield of synthetic wheats by ≥ 37%, 26%, and 50%, respectively. These studies suggest a presence of genetic variability among [italics]Aegilops[end italics] species that can be utilized in breeding wheat for HT and drought tolerance at anthesis; and combined stress of drought and high temperature on synthetic wheats are hypo-additive in nature.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / In this work were studied diverse physiological mechanisms, as the osmotic adjustment, photosynthesis, including gas exchange and chlorophyll fluorescence parameters, as well the oxidative responses in physic nuts submitted to different abiotic stresses as: salinity, drought and high temperature. The first experiment aimed to study the effects of increase of the NaCl concentrations (0, 25, 50, 75 and 100 mM) in the ions accumulate (Na+, Cl- and K+) and some growth variables, as well evaluate the water state and the principals solutes (organic and inorganic) involved on the osmotic adjustment of physic nuts plants under this stressful condition. Physic nuts plants showed sensibility to salt stress, presenting a reduction by 50% in dry matter from by 47 mM of NaCl concentration during 15-d. This sensibility should be due the leaf Na+ and Cl- high accumulation, associated a strong reduction in the K+ concentration, induced by high Na+ content. On the other hand, physic nuts plants were able osmotic adjust to salinity due a severe decrease on the osmotic potential and increase of leaf water state, principally in the higher NaCl levels. Of the solutes studied, was observed that salt ions (Na+ and Cl-) contributed with the most of the osmotic adjustment, while that the K+ contribution was decreased strongly by NaCl. The glycinebetaine compared to proline was more important to the osmotic adjustment of physic nuts leaves, as in the absence as in presence of different NaCl levels in the nutrient solution. The second experiment evaluated the resistance of photosynthetic apparatus of physic nuts plants submitted to different time of exposure (7-d and 14-d of treatment and 3-d of recovery) to salt stress (100 mM of NaCl).The changes caused on photochemistry activity and leaf gas exchange were evaluate by Na+ and Cl- accumulation and decrease of K+/Na+ ratio in the leaves. After 7-d of treatment was observed a major action of osmotic effects. However, after 14-d of treatment, the ionic effects caused by Na+ and Cl- excessive accumulation and by K+/Na+ ratio strong reduction in the leaves, caused permanent damages on the photosynthesis of physic nuts plants due as the stomatal limitations as non-stomatal ones. The third experiment aimed to study the comparative effects between the salt stress (50 mM of NaCl) and water stress (induced for PEG 6000), both with osmotic potential of â0.22 MPa on the photosynthesis, water relations and growth of physic nuts plants. The water stress effects induced for PEG in the leaf growth, electrolyte leakage and leaf gas exchange were more deleterious than by NaCl ones. In the both stresses was observed decrease in the leaf CO2 assimilation due the stomatal and non-stomatal limitations. However, the chlorophyll fluorescence parameters did not affect. The fourth experiment aimed to evaluate the relative contribution of organic and inorganic solutes on the osmotic adjustment of leaves and roots physic nuts plants in different water restriction levels. Of the solutes studied, the K+ and soluble sugar were the most involved in the osmotic adjustment as in the leaves as in roots. Others solutes as, Na+, Cl-, total amino acids and glycibetaine, also presented a effective role in the reduction of osmotic potential in both organs. On the other hand, the leaf proline content, although has increased significantly, was not sufficient to promote an effective participation of this amino acid in the osmotic adjustment of physic nuts plants. The same experiment aimed to observe the isolated and combined effects of water stress and high temperature on the photosynthesis and evaluate the oxidative defenses system in physic nuts plants. The photosynthetic apparatus was more sensitive to water stress than heat ones, been that the combination of them caused deleterious effects yet large in this complex. Additionally, the oxidative damages also were more marked in the combined stress. In general, the data shown that physic nuts plants, although present ability to adjust osmotically to salinity and drought, have their photosynthetic apparatus very affected in this stressful conditions. Even as, the defense system against oxidative damages appears has not been efficient in plants exposure at the drought and heat isolated and combined stresses / In this work were studied diverse physiological mechanisms, as the osmotic adjustment, photosynthesis, including gas exchange and chlorophyll fluorescence parameters, as well the oxidative responses in physic nuts submitted to different abiotic stresses as: salinity, drought and high temperature. The first experiment aimed to study the effects of increase of the NaCl concentrations (0, 25, 50, 75 and 100 mM) in the ions accumulate (Na+, Cl- and K+) and some growth variables, as well evaluate the water state and the principals solutes (organic and inorganic) involved on the osmotic adjustment of physic nuts plants under this stressful condition. Physic nuts plants showed sensibility to salt stress, presenting a reduction by 50% in dry matter from by 47 mM of NaCl concentration during 15-d. This sensibility should be due the leaf Na+ and Cl- high accumulation, associated a strong reduction in the K+concentration, induced by high Na+ content. On the other hand, physic nuts plants were able osmotic adjust to salinity due a severe decrease on the osmotic potential and increase of leaf water state, principally in the higher NaCl levels. Of the solutes studied, was observed that salt ions (Na+ and Cl-) contributed with the most of the osmotic adjustment, while that the K+contribution was decreased strongly by NaCl. The glycinebetaine compared to proline was more important to the osmotic adjustment of physic nuts leaves, as in the absence as in presence of different NaCl levels in the nutrient solution. The second experiment evaluated the resistance of photosynthetic apparatus of physic nuts plants submitted to different time of exposure (7-d and 14-d of treatment and 3-d of recovery) to salt stress (100 mM of NaCl).The changes caused on photochemistry activity and leaf gas exchange were evaluate by Na+ and Cl- accumulation and decrease of K+/Na+ ratio in the leaves. After 7-d of treatment was observed a major action of osmotic effects. However, after 14-d of treatment, the ionic effects caused by Na+ and Cl- excessive accumulation and by K+/Na+ ratio strong reduction in the leaves, caused permanent damages on the photosynthesis of physic nuts plants due as the stomatal limitations as non-stomatal ones. The third experiment aimed to study the comparative effects between the salt stress (50 mM of NaCl) and water stress (induced for xv PEG 6000), both with osmotic potential of â0.22 MPa on the photosynthesis, water relations and growth of physic nuts plants. T e water stress effects induced for PEG in the leaf growth, electrolyte leakage and leaf gas exchange were more deleterious than by NaCl ones. In the both stresses was observed decrease in the leaf CO2 assimilation due the stomatal and nonstomatal limitations. However, the chlorophyll fluorescence parameters did not affect. The fourth experiment aimed to evaluate the relative contribution of organic and inorganic solutes on the osmotic adjustment of leaves and roots physic nuts plants in different water restriction levels. Of the solutes studied, the K+ and soluble sugar were the most involved in the osmotic adjustment as in the leaves as in roots. Others solutes as, Na+, Cl-, total amino acids and glycibetaine, also presented a effective role in the reduction of osmotic potential in both organs. On the other hand, the leaf proline content, although has increased significantly, was not sufficient to promote an effective participation of this amino acid in the osmotic adjustment of physic nuts plants. The same experiment aimed to observe the isolated and combined effects of water stress and high temperature on the photosynthesis and evaluate the oxidative defenses system in physic nuts plants. The photosynthetic apparatus was more sensitive to water stress than heat ones, been that the combination of them caused deleterious effects yet large in this complex. Additionally, the oxidative damages also were more marked in the combined stress. In general, the data shown that physic nuts plants, although present ability to adjust osmotically to salinity and drought, have their photosynthetic apparatus very affected in this stressful conditions. Even as, the defense system against oxidative damages appears has not been efficient in plants exposure at the drought and heat isolated and combined stresses