Fisiologia Vegetal
URI permanente para esta coleçãohttps://locus.ufv.br/handle/123456789/185
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Resultados da Pesquisa
Item Caracterização fisiológica do gene homólogo ao transportador mitocondrial sucinato-fumarato (SFC1) de Arabidopsis thaliana em Solanum tuberosum(Universidade Federal de Viçosa, 2012-07-20) Brito, Danielle Santos; Nesi, Adriano Nunes; http://lattes.cnpq.br/3177036222169807Este trabalho objetivou caracterizar o papel fisiológico do transportador mitocondrial de sucinato-fumarato (mSFC1) em plantas de Solanum tuberosum. Para tanto, introduziu-se no genoma da batata um cassete contendo um fragmento do gene que codifica o SFC1 em Arabidopsis thaliana (AtmSFC1), cuja expressão foi reduzida pela técnica de RNA de interferência (RNAi). Por meio da técnica de PCR em tempo real a expressão do gene StSFC1, homólogo a AtmSFC1, foi quantificada. As linhas com menor expressão foram posteriormente cultivadas em casa de vegetação para as análises de trocas gasosas, parâmetros de fluorescência da clorofila a e de crescimento, bem como análises bioquímicas. Os resultados obtidos revelaram que, apesar de as linhas transgênicas apresentarem uma maior biomassa na parte aérea, a redução na expressão de StSFC1 não proporcionou nenhuma alteração fisiológica e bioquímica significante nas variáveis analisadas em folhas. Já em tubérculos, observou-se um decréscimo significativo nos níveis de amido nas linhas transgênicas, em relação ao controle. Os resultados obtidos sugerem que o gene SFC1 em batata parece possuir uma função fisiológica minoritária em tecidos autotróficos como folhas. Entretanto, em tecidos heterotróficos, como tubérculos em desenvolvimento, a ausência do transportador leva a alterações metabólicas que podem limitar o acúmulo e/ou a síntese de amido nos amiloplastos.Item The role of 2-oxoglutarate dehydrogenase during water deficit and recovery in Arabidopsis thaliana(Universidade Federal de Viçosa, 2018-02-18) Vargas, Jonas Rafael; Nesi, Adriano Nunes; http://lattes.cnpq.br/9028236058582130To deal with periods of water limitation, plants use different mechanisms. Among them, stomatal closure and proline accumulation are the major ones during stress exposure. After stress, proline is degraded releasing glutamate in the mitochondrial matrix. The glutamate is metabolized by different metabolic pathways and may: (i) participate in nitrogen metabolism, being used for ammonia assimilation under the action of enzymes like Glutamine synthetase (GS) and Glutamine 2-oxoglutarate aminotransferase (GOGAT), (ii) being reintroduced in the tricarboxylic acid cycle (TCA cycle) as succinate via GABA shunt activity or (iii) enter the TCA cycle as 2-oxoglutarate (2-OG) when undergoing the action of the enzyme glutamate dehydrogenase. 2-OG is converted into succinyl-CoA by the action of the enzyme 2-oxoglutarate dehydrogenase (2-OGDH). In order to understand the importance of 2-OGDH during water deficit and stress release, two T-DNA insertion lines with low expression of the E1 subunit of 2-OGDH encoding gene, e1-ogdh1.1 and 1.2 were submitted to drought conditions for 22 days and then, after rewatering, three days of recovery period. This work revealed that the TCA cycle goes through downregulation in such a way that allows plants to cope with water deficit and that following recovery period its activity is reestablished. Furthermore, it was observed that the reduction in the activity of 2-OGDH did not demonstrate great impact in the metabolism during the implementation and recovery of water deficit. Keywords: abiotic stress; metabolism; proline; TCA cycle; 2-OGDH.Item Functional analysis of the thioredoxin system during seed germination in Arabidopsis thaliana(Universidade Federal de Viçosa, 2019-03-29) Nascimento, Carolina Pereira; Nesi, Adriano Nunes; http://lattes.cnpq.br/3331610618877369A series of processes occurs during seed formation, including remarkable changes from early development to the end of germination. The changes associated with processes initiated mainly after seed imbibition are usually characterized by extensive changes in redox state of seed reserve proteins and of pivotal enzymes for protein mobilization and usage. Such changes in redox state are often mediated by Thioredoxins (Trxs), which are protein oxiredutases capable of catalyzing the reduction of disulfide bonds in target proteins, thereby regulating their structure and function. Here, we analyzed the previously characterized mutants of NADPH-dependent Trx reductase A and B (ntra ntrb), two independent mutant lines of mitochondrial thioredoxin o1 (trxo1) and two mutant thioredoxin h2 (trxh2) mutant lines. Our results indicate that plants deficient for the NADPH-dependent thioredoxin system are able to mobilize their reserves, but at least partially fail to use these reserves during germination, thereby leading to lower availability of energy substrates than wild type seeds. Trx mutants also show decreased activity of regulatory systems needed to maintain cellular homeostasis. Moreover, we observed reduced respiration in mutant seeds and seedlings, which in parallel with an impaired energy metabolism, disrupts core biological processes responsible for proper germination and early development of Trx mutants. In conclusion, the results suggest that the lack of thioredoxin induces a substantial adaptation in seeds and seedlings, which undergo a metabolic reprogramming to adapt to a new redox state.Item On the function of guard cell transporters in the response of Arabidopsis thaliana to changes in CO2 and water availability(Universidade Federal de Viçosa, 2019-02-25) Luz, Luana Moraes da; Araújo, Wagner Luiz; http://lattes.cnpq.br/7249150408731894Plants represent the major sources worldwide of human foods and livestock feeds. Thus, future food security will clearly depend on how plant species respond to global environmental changes. Increases in carbon dioxide concentration [CO 2 ] in the atmosphere and fluctuations in rain patterns are amongst the main climate changes affecting crop yield. Notably, crop yield decreases under drought, yet elevated [CO 2 ], when associated with drought, may mitigate the negative effects of drought. Among the physiological effects of elevated [CO 2 ], partial stomatal closure and synergistically increased water use efficiency (WUE), leading to higher growth, are usually observed. However, either higher growth or survival of plants under high [CO 2 ] will directly depend on the degree of water availability into the soil. Water losses are controlled directly by the stomata, functionally specialized microscopic pores in leaf surface, that regulates the flow of gases between plants and atmosphere. Accordingly, stomata are the key entry point for CO 2 assimilation and water losses, controlling the essential exchange of CO 2 and H 2 O with the environment in land plants. Stomatal movements occur in response to the activation and inactivation of membrane proteins present in the guard cells. The relationship between stomatal aperture and photosynthesis/transpiration is linear over a wide range of environmental conditions. Thus, a better understanding of stomatal regulation by environmental stimuli represents an important step for developing plants in which WUE and photosynthesis capacity are optimized, once the maintenance of photosynthesis, one of the main events of the primary metabolism, and its capacity may directly influence crop yield. This thesis is largely focused on the role of proteins involved in the stomatal movements in response to changes in both [CO 2 ] and water availability. To this end, two stomatal proteins in Arabidopsis thaliana, namely the slow-type anion channel (SLAC1) and the ATP-binding cassette B14 transporter (ABCB14) were used to further investigate the duality of the efficiency between the CO 2 influx and the water efflux by the stomata under elevated [CO 2 ] and water limitation. SLAC1 is considered a key protein for stomatal closure in response to drought and [CO 2 ], while ABCB14 is the sole influx transporter of malate of the guard cell and is only responsive to increased CO 2 . The main goal of this study is to increase our understanding of the stomatal regulation in response to a realistic future climate change scenario using a metabolic, physiological, anatomical and molecular characterization of mutant plants with opposite stomatal responses. First, our results demonstrated that slac1 mutant plants are seemingly constantly under stress effect, regardless of the water restriction, since to sustain the increased stomatal conductance (g s ) slac-1 mutant plants display an elegant metabolic reprogramming that is apparently crucial for growth survival under moderate water limitation. Here we further hypothesized that ABCB14 may play an important role while allowing a modest stomatal opening that may be essential for continuity of gas exchanges under high [CO 2 ] conditions. In the second part, it was demonstrated that despite the reductions in the stomatal opening, the absence of a functional guard cell ABCB14 protein does not compromise overall photosynthetic activity and growth and that an exquisite metabolic and genetic regulation occurs in guard cells likely compensating, at least partially, the functional lack of abcb14. The data obtained here are discussed in the context of the role of each guard cell transport both generally to guard cell and photosynthetic metabolism and specifically with respect to its function in the regulation of stomatal aperture.Item Importância fisiológica do catabolismo de lisina durante estresse salino em Arabidopsis thaliana(Universidade Federal de Viçosa, 2017-12-15) Neves, Tárik Galvão; Araújo, Wagner Luiz; http://lattes.cnpq.br/0345441921253142Solos salinos, aquele em que as concentrações de sal excedem à 10 mM de cloreto de sódio (NaCl), geram danos significativos que afetam o crescimento e desenvolvimento de cultivos agrícolas. A ocorrência de solos salinos é comum em regiões áridas e semi-áridas. No entanto, áreas agricultáveis podem sofrer com a salinização onde, em conjunto com um sistema de drenagem natural ou artificial ineficaz, a água de irrigação, ainda que de boa qualidade, pode gerar o acúmulo de sal na rizosfera. Os impactos em plantas causados pelo excesso de sal nos solos culminam em (i) estresse osmótico que reduz o potencial hídrico do solo, dificultando a obsorção de água pelas raízes, e (ii) desbalanço iônico celular caso altas concentrações de Na + ou Cl - forem absorvidos. Em conjunto, os danos causados pela presença de altas concentrações de NaCl na solução do solo diminuem a absorção de água e nutrientes, interferindo em processos metabólicos importantes como fotossíntese e respiração. Cumpre ressaltar que as mitocôndrias de plantas desempenham deversas funções importantes para o metabolismo vegetal e, em condições de estresse salino, a geração de energia mediada pelo ciclo dos ácidos tricarboxílicos (TCA) e pela cadeia transportadora de elétrons mitocondrial (mETC) são afetados pelo déficit de carboidratos provenientes da fotossíntese. Nesse contexto, vias alternativas da respiração são induzidas fornecendo elétrons para mETC e compostos intermediários para o ciclo do TCA. Estudos recentes mostram que a degradação de proteínas e catabolismo de aminoácidos podem gerar incrementos nas taxas respiratórias em condições de escassez de carbono. Tanto a biossíntese quanto o catabolismo de lisina apresentam uma estreita ligação com o metabolismo energético em especial com o ciclo do TCA. Dessa forma, o presente trabalho buscou investigar o papel da lisina nas respostas fisiológicas e metabólicas em resposta ao estresse salino. Para tanto, os impactos ocasionados ao crescimento e metabolismo foram analisados em mutantes na biossíntese de lisina fornecendo evidências fenotípicas e fisiológicas da importância de lisina para uma maior tolerância ao estresse salino em Arabidopsis thaliana. Plântulas de Arabidopsis com redução da atividade da enzima L,L-diaminopimelato aminotransferase (dapat) mostraram uma maior sensibilidade ao estresse salino quando comparadas ao seu tipo selvagem, apresentando baixa germinação de sementes em todos tratamentos com estresse salino, enquanto mutantes para a dihidropicolinato sintase (dhdps-2) apresentaram maior tolerância ao estresse salino. Além disso, foi observado em todos genótipos reduções progressivas nos valores da eficiência fotoquímica máxima do fotossistema II (F v /F m ) ocorrendo de forma mais acelerada quando submetidas a 150 mM de NaCl. Concomitantemente, diminuições nos teores de clorofila total e na razão clorofila a / clorofila b foram observadas. Não obstante, o estresse salino induziu o acúmulo de aminoácidos totais, bem como diminuições nas concentrações de proteínas totais e amido. Assim, é plausível sugerir que outros compostos, que podem atuar como osmorreguladores, tiveram papel importante nas respostas aqui observadas. Os resultados apresentados demonstram a importância de lisina como um substrato alternativo para manutenção da homeostase celular em condições de estresse salino.Item The influence of the Target of Rapamycin (TOR) on starch metabolism in Arabidopsis thaliana(Universidade Federal de Viçosa, 2016-02-22) Araujo, Elias Feitosa; Caldana, Camila; http://lattes.cnpq.br/4607484418104157Plant growth and development are maintained by a complex network controlled by environmental factors including the availability of water, light and nutrients and by several signaling pathways. One of the most important signaling pathways, conserved in eukaryotes, is the kinase Target of Rapamycin (TOR). Various lines of evidence point out that TOR plays a fundamental role in carbon and nitrogen balance, acting as an essential regulator on central metabolism by controlling growth and biomass production. Starch is the major form of carbon storage and its content is negatively correlated with growth. Transgenic lines with reduced expression of TOR gene or components of the TOR complex present a clear starch excess phenotype. However, it remained to be elucidated whether the accumulation of starch is due to increased synthesis, impaired degradation or both. In this work, Arabidopsis seedlings treated with the specific ATP-competitive inhibitor of TOR kinase AZD-8055 showed a starch excess phenotype right after 4 hours of treatment and the accumulation of starch was proved to be due to an augmentation in the rate of starch synthesis. Furthermore, TOR- inhibited plants presented an average increase of 20-30% in their starch content at the end of day when compared to control. Metabolite profiling analysis showed that TOR– inhibited plants exhibited broad changes in the levels of sucrose, fructose, glucose, maltose, mannose and orthophosphate, which are associated directly or indirectly with starch metabolism. In addition, a correlation between the amount of mannose, orthophosphate and increased starch content was noticed in AZD-treated plants. Gene expression analysis of AGPase subunits showed significant changes only from 18 and 24h after treatment. Although TOR inhibited plants displayed higher content of the active form of AGPase (monomer), enzymatic activity assays revealed that changes in AGPase activity might occur as secondary effect of TOR inhibition and might be not related to the starch excess phenotype observed 4 hours after AZD-treament. Since several enzymes related to starch metabolism are subject to redox regulation, the levels of glutathione were measured to verify the redox environment of the cells. TOR- inhibited plants showed changes in the pools of glutathione, mainly in its reduced form, and the redox state of the cells tended to be more reduced. Together, these results indicate the participation of TOR signalling on starch metabolism but the mechanistic behind this process need further studies.Item Functional analysis of mitochondrial proteins in Arabidopsis thaliana(Universidade Federal de Viçosa, 2016-08-26) Brito, Danielle Santos; Nesi, Adriano Nunes; http://lattes.cnpq.br/3177036222169807Mitochondrial carrier family (MCF) proteins catalyze the specific transport of various substrates, such as nucleotides, amino acids and cofactors. Although some of the mitochondrial transporters have been identified, many of these proteins have not yet been completely characterized. Likewise, the proteic machinery and mechanisms involved in the mitochondrial alternative respiration is still not well known. In this context, this work first presents a study of a previously identified but uncharacterized mitochondrial transporter AtSFC1, a potential succinate/fumarate carrier. Hence, to obtain the biochemical role of AtSFC1, we carried out substrate specificity and investigated its physiological function using 35S antisense transgenic lines in Arabidopsis thaliana. Briefly, the functional integration of AtSFC1 in the cytoplasmic membrane of intact Escherichia coli cells reveals a high specificity for a citrate/isocitrate in a counter exchange mode. Additionally, we discussed the potential role for AtSFC1 in the provision of intermediates of tricarboxylic acid cycle to provide carbon and energy to support growth in heterotrophic tissues. In the second part of this thesis, we investigated the function of alternative electron donors to the mitochondrial electron transport chain (mETC) during carbon deprivation as well as after the supply of amino acids. The breakdown products of branched chain amino acids can provide electrons to the mETC via the ETF/ETFQO (electron transfer flavoprotein: flavoprotein ubiquinone oxidoreductase) complex. This system is located in the mitochondria and induced at the level of transcription during stress situations. Thus, in order to obtain a comprehensive picture of how alternative respiration pathway interacts with other pathways and adjust to different cellular and metabolic requirements, we performed metabolic and physiological approaches using Arabidopsis cell culture ETFQO T-DNA insertion mutants. The results discussed here support that the ETF/ETFQO system is an essential pathway able to donate electrons to the ubiquinone pool. In addition, the behavior of the respiratory complexes suggest new electrons entry points, which must be elucidated.Item Molecular and metabolic responses associated with the lack of autophagy following energy deprivation in Arabidopsis thaliana(Universidade Federal de Viçosa, 2016-07-18) Barros, Jessica Aline Sousa; Araújo, Wagner Luiz; http://lattes.cnpq.br/8783435505580467The oxidation of carbohydrate in mitochondria is the primary energy source for cellular metabolism. However, during energy-limited conditions alternative substrates are required to support respiration. The oxidation of amino acids plays a key role in this process by generating electrons that can be transferred to mitochondrial electron transport chain via the electron transfer flavoprotein/ ubiquinone oxireductase (ETF/ETFQO) system. Compelling evidence has demonstrated the close association of autophagy in providing alternative substrates for power generation under carbohydrate-limited conditions; however, how and to which extent autophagy and primary metabolism interact to support respiration remains unclear. To obtain a comprehensive picture of the metabolic importance of autophagy during development and extended darkness Arabidopsis thaliana mutants with impairments in autophagy were used. atg mutants showed reduction of growth and seed production. Following extended darkness atg mutants were characterized by early signs of senescence as well as decreased chlorophyll content and maximum photochemical efficiency of PSII (Fv/Fm). Metabolite profile of dark-treated leaves revealed an extensive metabolic reprogramming in which increases in amino acids contents were partially compromised and thus limiting their utilization as substrate to sustain respiration in atg mutants. Additionally, transcript levels of genes involved in alternative pathways of respiration, amino acid catabolism, and chloroplast vesiculation (CV) were up-regulated in atg mutants. Our results thus suggest that autophagy contributes to energy availability by supplying amino acids for alternative pathways of respiration. Furthermore, our finding demonstrated the potential role of CV as a compensatory protein degradation pathway under C-limiting conditions when autophagy is impaired .Item Functional characterization of the mitochondrial adenine nucleotide transporter (ADNT1) in Arabidopsis thaliana under dark-induced senescence(Universidade Federal de Viçosa, 2013-02-28) Pereira, Paula da Fonseca; Nesi, Adriano Nunes; http://lattes.cnpq.br/6419216674405263Unlike other ADP/ATP carriers, ADNT1 is the only one that mediates an antiport of ATP, AMP, and, to a lesser extent, ADP and the corresponding deoxyadenine nucleotides. Previous work observed that ADNT1 expression is much stronger in root tips and senescing tissues. Considering the high expression of ADNT1 in senescing tissues, we have investigated the role of ADNT1 during the process of dark-induced senescence. Under these conditions, Arabidopsis thaliana mutants deficient in the expression of ADNT1 transporter displayed a similar, yet milder, early onset of senescence as evidenced both by the visual phenotype of plants following growth in extended periods of darkness and the loss of chlorophyll and photosynthetic competence. The extended dark treatment led in general to a more rapidly decline in the mutants than in the wild type in the levels of sucrose and protein. By contrast, the levels of total amino acids and TCA cycle intermediates malate, fumarate and isocitrate generally increased significantly in the mutants at the end of dark treatment. The NADH/NAD + and NADPH/NADP+ ratios also increased in mutants in comparison to the wild type with progression of the darkness. Additionally, the mutant plants exhibited symptoms of early senescence in comparison to the wild type even under optimal conditions. Altogether the data obtained demonstrate that ADNT1 is not functionally redundant to the previously characterized ADP/ATP carriers, especially during carbon starvation and reinforce the potential function for ADNT1 in the provision of energy which is required to support growth in heterotrophic plant tissues.Item Alterações fisiológicas causadas pelo arsênio, genotoxidade e importância do mecanismo mismatch repair no reparo do DNA em Arabidopsis thaliana(Universidade Federal de Viçosa, 2013-04-02) Barbosa, Alice Pita; Loureiro, Marcelo Ehlers; http://lattes.cnpq.br/3328314264518039O arsênio (As) é um elemento não só tóxico, mas também altamente genotóxico aos seres vivos. Muitas lacunas precisam ser preenchidas com relação aos processos causadores de toxidade do As em plantas, bem como os mecanismos de tolerância e sensibilidade a este metalóide. Para isso, plantas de Arabidopsis thaliana (WT, mutantes msh2 e transgênicas repórteres em mutações) e Allium cepa foram expostas a 0, 2, 8 e 16 mg As L -1, durante cinco dias, em sistema hidropônico ou em meio de cultura. As plantas acumularam grandes teores de As nas raízes e apresentaram elevado fator de translocação para a parte aérea, e também alterações no acúmulo de nutrientes. Os sintomas visuais se intensificaram com o aumento da concentração de As na solução nutritiva. As raízes adquiriram coloração escura e aspecto gelatinoso, danificado e aumento no comprimento e densidade dos pêlos; a parte aérea apresentou aumento dos teores de antocianinas e sinais de senescência precoce, bem como alterações na espessura de tecidos. O estresse oxidativo e a redução dos teores de fósforo foram apontados como os principais efeitos do As capazes de causar toxidez, evidenciando os danos indiretos deste elemento no organismo. Foram verificadas importantes alterações fotossintéticas, bem como indícios de danos ao processo de respiração celular devido o aumento da expressão de genes codificantes de oxidases alternativas. Também foram observadas alterações nos teores de açúcares em folhas jovens, maduras e raízes. O As promoveu fragmentação do DNA nos ápices radiculares de A. cepa e aumento das taxas de mutação pontual e de recombinação-não homóloga em A. thaliana. O significativo aumento da expressão dos genes msh2 e msh7, codificadores de enzimas-chave do processo mismatch repair, que realiza o reparo de bases danificadas ou erroneamente inseridas no DNA, sugeriu a importância deste mecanismo no combate à genotoxidade do As em A. thaliana. Isso foi confirmado pela maior sensibilidade observada nas plantas mutantes msh2 ao As, detectada visualmente via aumento da peroxidação de lipídios. Observou-se inibição da atividade da protease caspase-3, associada ao processo de morte celular programada, reforçando a capacidade de inibição da atividade enzimática pelo As.