Estudio y purificación de peroxidasas implicadas en la lignificación de plantas basales
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http://hdl.handle.net/2183/11558Coleccións
- Teses de doutoramento [2089]
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Estudio y purificación de peroxidasas implicadas en la lignificación de plantas basalesAutor(es)
Director(es)
Novo Uzal, EstherMerino de Cáceres, Fuencisla
Pomar Barbeito, Federico
Data
2013Centro/Dpto/Entidade
Universidade da Coruña. Departamento de Bioloxía Celular e MolecularResumo
[Abstract] The colonization of the terrestrial environment by plants has led to the development of different mechanisms which allow the survival in a hostile environment. One of these adaptations has been the occurrence of lignins and lignification process that gave plants the necessary improvement to stay upright and an efficient water transport. The first organisms to colonize land were non-vascular plants, these species were structurally simple but had the ability to adapt to new environment because of their high resistance to stress. These first plants were classified as bryophytes and divided into three groups; liverworts, mosses and hornworts. Bryophytes do not have a vascular system and most of them do not have lignin. However, there are some exceptions such as the liverwort Marchantia polymorpha which possesses guaiacyl and syringyl lignins. The moss Physcomitrella patens also represents an exception, even though it does not contain lignins, it has the ability to oxidize coniferyl and sinapyl alcohols, which are linked to the biosynthesis of guaiacyl and syringyl units. The earliest vascular plants were pteridophytes, which contain predominantly guaiacyl lignins in their cell wall. However, numerous exceptions have been recently found, such as Selaginella martensii, which contains 70% syringyl lignins with a lower proportion of guaiacyl units, a typical feature of angiosperms. Furthermore, this production of syringyl lignins occurs through a novel mechanism of synthesis, in which two new enzymes have been developed to supplement the lack of ferulate 5-hydroxylase and caffeic acid 3-O-methyltransferase, the necessary enzymes for the production of syringyl lignins. These features make the study of these three species of great interest in the syringyl lignin biosynthesis. Moreover, basic peroxidases have been characterized as responsible for the oxidation of sinapyl alcohol in several works. Therefore, it is necessary a deep study of basic peroxidases present in these three non angiosperms species. By means of histochemistry, M. polymorpha showed a colocalization of peroxidase activity and lignins in its rhizoids. Furthermore, isoelectrofocusing analysis determined a coincidence between some basic peroxidases with the peroxidase of Z. elegans responsible of lignification. Therefore, peroxidases of M. polymorpha were purified using a three-step protocol which included ammonium sulfate precipitation, adsorption chromatography on Phenyl Sepharose and cationic exchange chromatography on SP Sepharose. A fraction enriched in basic peroxidases presented the ability to oxidize coniferyl and sinapyl alcohols and ferulic acid. These features support the fact that these peroxidases are involved in lignification of this species. P. patens is a species without lignins, but it presents phenolic compounds known as lignin-like. The purification of peroxidases was achieved in a three-step protocol, like in the purification of M. polymorpha. This process enabled us to isolate a peroxidase (PpPrx) with an isoelectric point of 10.04 and a molecular weight of 36.5 kDa. This peroxidase was able to oxidize both coniferyl and sinapyl alcohols. In addition, the kinetic analysis showed a high catalytic efficiency against coniferyl alcohol, similar to other peroxidases involved in lignification of other species. To check how this enzyme acts against several abiotic stresses, plants of P. patens were treated in liquid culture with compounds that activate the signaling pathway of ABA (abscisic acid, sodium chloride and mannitol), a compound involved in the response to pathogens (salicylic acid) and a compound that activates the path of oxidative stress (hydrogen peroxide). The culture medium pH, total peroxidase activity, protein and peroxidase patterns and biosynthesis of phenols were analysed after the different treatments. Interestingly, the purified peroxidase PpPrx was induced with sodium chloride and hydrogen peroxide treatments, whereas it was inhibited by abscisic acid and mannitol treatments. In addition, the rhizoid secretome of P. patens was studied in two stages of development in order to check which proteins were secreted into the culture medium by rhizoids under normal conditions. A total of 47 proteins in cultures of 7 days and a total of 67 proteins at 28 days were identified. These proteins were clustered into different groups based on their involvement in diverse processes such as cell wall modification, senescence or regulation. In S. martensii, histological sections showed a colocalization of peroxidase activity and lignin. Whereas guaiacyl lignins were located in the xylem, syringyl lignins were located in the epidermis and sub-cortical epidermis. a purification protocol was performed in three steps as described above for M. polymorpha. Three basic peroxidases were obtained; one semipurified (SmaPrx1) and two proteins purified to homogeneity (SmaPrx2 and SmaPrx3). SmaPrx1 fraction showed a basic isoelectric point and molecular weight between 35 and 45 kDa, typical of class III peroxidases. Moreover, activities against several substrates suggest that these peroxidases are involved in lignification. The molecular mass for SmaPrx2 and SmaPrx3 was calculated to be 36.3 kDa and 45.6 kDa, respectively, according to MALDI-TOF/TOF. Both enzymes show a typical peroxidase UV-visible spectrum with a Soret peak at 403nm for SmaPrx2 and 404nm for SmaPrx3. The specific activities showed against several substrates and the kinetic parameters suggest SmaPrx2 and SmaPrx3 have specific roles in cell wall formation and especially in lignin biosynthesis. Several peptides from tryptic digestion of both peroxidases were identified through MALDI-TOF MS/MS. The presence in these peptides of structural determinants typical of syringyl peroxidases indicates these proteins show no structural restrictions to oxidize syringyl moieties. These data, along with the in vitro capacity of using sinapyl alcohol as substrate and the low KM in the µM range suggest these two peroxidases may be responsible for the oxidation of syringyl monolignols that leads to syringyl lignin biosynthesis.
Palabras chave
Peroxidasas
Pared celular
Plantas
Pared celular
Plantas
Dereitos
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