Estudio de la regulación redox en tejidos fotosintéticos y no fotosintéticos de Arabidopsis thaliana.
- Autores: Julia Ferrández Navarro
- Directores de la Tesis: Francisco Javier Cejudo Fernández (dir. tes.), María Cruz González García (dir. tes.)
- Lectura: En la Universidad de Sevilla ( España ) en 2013
- Idioma: español
- Número de páginas: 258
- Tribunal Calificador de la Tesis: Francisco Javier Florencio Bellido (presid.), Lola Peñarrubia Blasco (secret.), Luis Carlos Romero González (voc.), J. Antonio Bárcena Ruiz (voc.), Eevi Rintamäki (voc.)
- Materias:
- Enlaces
- Tesis en acceso abierto en: Idus
- Resumen
The main objective of this thesis was to elucidate the processes regulated by NTRC in plastids of both photosynthetic and non-photosynthetic tissues and the contribution of this enzyme to plant development in Arabidopsis thaliana. To this end, the following specific objectives were addressed:
1. Identification and characterization of proteins interacting with NTRC in photosynthetic and non-photosynthetic tissues.
2. To stablish the function of NTRC in the global redox homeostasis in Arabidopsis and in the development of heterotrophic tissues.
Redox regulation based on dithiol-disulfide interchange of key Cys residues of regulatory enzymes is an essential mechanism of signaling and regulation that plays a central role in the response of plants to enviromental estimuli. Redox regulation has become increasingly important in the control of chloroplast metabolism, in which NADPH thioredoxin reductase C (NTRC) plays an important role (Pérez-Ruiz et al. 2006). NTRC is a chloroplast-localized NTR with a joint thioredoxin domain that conjugates both activities to efficiently reduce 2-Cys-Prx using NADPH as source of reducing power (Pérez-Ruiz et al., 2006). Recently, we have found that NTRC is also located in plastids of non-photosynthetic tissues (K. Kirchsteiger, Doctoral Thesis), which lack photochemical reactions so that their redox regulation depends exclusively on NADPH produced from sugars. Thereby, NTRC may play an essential role in maintaining redox homeostasis in plastids of both photosynthetic and nonphotosynthetic tissues. In this regard, the fact that an Arabidopsis NTRC knockout mutant causes a much more severe phenotype than that of the double knock down mutant of 2-Cys-Prx (Pulido et al., 2010), points out that NTRC must have additional functions to the reduction of 2-Cys PRX. Some of these functions, including the regulation of starch (Michalska et al., 2009) or aromatic amino acid and auxin synthesis (Lepisto et al., 2009), have already been identified. However, a better understanding of the role of NTRC in maintaining the redox homeostasis of plastids of photosynthetic and non-photosynthetic tissues could be achieved if more were known about their target proteins. To this end, we used three methodologies couple with proteomics: affinity chromatography with monocisteinic NTRC, immunoprecipitation and fluorecescent gel electrophoresis. By application of the three methods, we isolated 52 targets in chloroplasts and 33 in amyloplasts, 26 of them not previously recognized by other redoxins. The potential NTRC targets function in a range of processes: metabolism of carbohydrates, amino acids, lipids, nucleotides, coenzymes; protein folding; detoxification; defense; energy; and translation. These results remarked the importance of redox regulation not only in chloroplasts, but also in non-green plastids and raised the possibility that NTRC might be acting as a regulatory link, whereby functions of chloroplasts and non-green plastids, such as amyloplasts, are integrated to harmonize the growth of the different organs of the plant. To test this possibility we generated transgenic Arabidopsis plants with leaf- or root-specific expression of NTRC in the ntrc mutant, thus recovering the redox homeostasis exclusively in chloroplasts or in amyloplasts, respectively. The analyses of these plants revealed that redox regulation of chloroplast is essential and necessary for the growth and development of both photosynthetic and non-photosynthetic tissues, not only because chloroplasts are the source of energy and nutrients, but also because exert a crucial role in signaling to the non-green plastids. As NTRC deficiency causes impairment of auxin synthesis (Lepistö et al., 2009) and lateral root formation, a phenotype clearly related to auxin, one of these signals might be auxins. Transgenic lines expressing an auxin reporter gene, ProDR5- GUS, confirmed the lower auxin content of the ntrc mutant and suggested that NTRC is involved in auxin homeostasis. Nitrilase 1 (NIT1), an enzyme involved in auxin biosynthesis, was found among the NTRC targets in photosynthetic tissues. Here we show that the ntrc mutant has impaired the redox state of NIT1 and reduced sensitivity to IAN, but not to IAA, thus supporting the idea that the function of NIT1 is regulated by NTRC and proposing that this link might contribute to the auxin synthesis. In addition, we further show that auxin transport is perturbed in ntrc mutant plants.
Additionally, it is well known the existence of a link between light and enzyme activity through chloroplast TRXs. By contrast, a little role of NTRC was postulated in this regulation (Meyer et al., 2009). However among the chloroplast NTRC targets we found proteins as TK, an enzyme involved in both Calvin cycle and the oxidative pentose phosphate pathway. While the activity of TK is positively affected by TRX f, NTRC is affecting negatively. Interestingly, this deactivation is dependent on the catalytic activity of NTRC, suggesting that NTRC has the ability to oxidize thiol groups. Moreover, our in vitro results suggested the existence of a crosstalk between TRX f and NTRC that controls the activation/deactivation of TK. These findings open the possibility that NTRC might play a new role in redox homeostasis, participating in the deactivation of light-enzymes during the night.
