Ciências Biológicas e da Saúde
URI permanente desta comunidadehttps://locus.ufv.br/handle/123456789/3
Navegar
2 resultados
Resultados da Pesquisa
Item The role of NAD+ compartmentation and dynamics in Arabidopsis thaliana(Universidade Federal de Viçosa, 2020-02-19) Araujo, Elias Feitosa; Nesi, Adriano Nunes; http://lattes.cnpq.br/4607484418104157Nicotinamide adenine dinucleotide (NAD+) is a fundamental coenzyme required to regulate plant central metabolism and redox status homeostasis. Plants produce NAD+ via de novo and salvage pathways. Despite early steps of the two pathways occur in different subcellular compartments, de novo in chloroplasts and salvage in cytosol, both pathways produce the final product NAD+ exclusively in the cytosol. Thus, NAD+ must be imported into organelles to drive biological processes inside them. In Arabidopsis thaliana, three genes have been identified as NAD+ carriers, namely NDT1 (At2g47490) and NDT2 (At1g25380), both targeted to the inner mitochondrial membrane, and the peroxisomal transporter PXN (At2g39970). The previous functional characterization of NDT1, NDT2 and PXN have revealed their importance for seed production and quality, seedling establishment, photosynthesis, metabolism, stomatal density and stomatal conductance. To extend our knowledge on the importance of NAD+ dynamics, we used mutants for NAD+ transport and focused in comprehend: (1) how altered NAD + distribution affects stomatal development in cotyledons, (2) the crosstalk between NAD+ transport and elevated atmospheric CO2 concentration and (3) NAD + dynamics in vivo under different environmental cues. The results indicate that NAD negatively regulates stomatal development in cotyledons of Arabidopsis. Seedlings with reduced expression of mitochondrial (NDT1 and NDT2) and peroxisomal (PXN) NAD+ transporter genes displayed reduced numbers of stomata lineage cells and reduced stomatal density. Furthermore, cotyledons of wild- type seedlings treated with exogenous NAD+ and cotyledons of mutant plants with reduced NAD+ breakdown capacity also exhibited reduced stomatal number. Impaired NAD+ transport and the exogenous NAD+ feeding were further associated with the induction of abscisic acid (ABA)-responsive genes. Additionally, NAD+ feeding of aba- 2 and ost1 seedlings, impaired in ABA synthesis and ABA signaling, respectively, did not impact on stomatal number, whereas the inhibition of ABA synthesis rescued the stomatal phenotype in NAD+ carrier mutants. Moreover, in vivo measurement of ABA dynamics in seedlings of an ABA-specific optogenetic reporter - ABAleon2.1 treated with NAD+ showed increases in ABA content, suggesting that NAD+ impacts on stomatal development through ABA synthesis and signaling. The results demonstrate that intracellular NAD+ homeostasis is essential for normal stomatal development, and provide a link between central metabolism and developmental plasticity. Posteriorly, NAD+ carrier mutants were grown under elevated CO2 concentrations and overall results showed that, under high CO2 , the mutants displayed reductions in total biomass and leaf number compared to the control under ambient CO2 . Furthermore, higher levels of photorespiratory intermediates such as glutamate and glycine were found in the mutant lines under elevated CO2 . Moreover, mutant lines produced much less seeds than wild-type plants regardless of CO2 concentration, demonstrating that NAD+ compartmentalization is fundamental during reproductive phase in both ambient and high CO2 concentration. With the aim to deeper study NAD + dynamics, we transformed wild-type and a ndt1 mutant line with the Peredox-mCherry sensor that permits the measuring of cytosolic NADH dynamics in vivo. The cytosol of ndt1 mutant lines presented higher levels of NADH/NAD + compared to the control line. Additionally, increased cytosolic levels of NADH/NAD+ in leaves of ndt1 mutants was observed upon the exogenous feeding of sugars and tricarboxylic acid (TCA) cycle intermediates. Surprisingly, light, mitochondrial electron transport chain (mETC) inhibitors and oxygen deprivation treatment did not show significant differences in cytosolic NADH/NAD+ content in ndt1 mutants compared to wild-type. The results provide evidence of disruption of NAD+ balance among organelles in ndt1 mutants and show the power of this technique to follow NADH dynamics in vivo. Collectively, the data presented provide different inputs to show that, not only NAD+ metabolism, but also NAD+ distribution across organelles, are fundamental to drive essential biological processes in plants. Furthermore, this study proposes a direct link between central metabolism and early developmental process such as stomatal development. Keywords: Nicotinamide adenine dinucleotide. NAD+ transport. Metabolism. Development.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.