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Universidade do Minho (2009)

Molecular mechanisms associated to thermotolerance in plants

Correia, J

Titre : Molecular mechanisms associated to thermotolerance in plants

Auteur : Correia, J.

Université de soutenance : Universidade do Minho

Grade : Tese de doutoramento em Ciências 2009

Résumé
One of the most typical abiotic stresses encountered by plants is extreme temperatures. High temperature leads to a series of morphological, physiological and molecular alterations that adversely affect plant growth and productivity. Acquisition of thermotolerance is largely controlled through molecular mechanisms based on the activation and regulation of specific stress‐related genes. The elucidation of these gene/protein functions will give insights on the various mechanisms of plant response to heat stress, providing useful information to improve plant thermotolerance. The present work aims to contribute for the understanding of the molecular mechanisms that are responsible for plant adaptation to heat stress. Two species, Populus euphratica Oliv. and Arabidopsis thaliana L., were used as models due to the latest development of genomic and molecular biology resources and tools for both plants. P. euphratica is naturally found under severe conditions such as extreme temperatures (‐45°C to +54°C), high soil salinity and drought. The physiological response of P. euphratica cultured cells was evaluated at different temperatures. Contrasting with its innate enhanced tolerance to extreme temperatures, the in vitro system did not present an outstanding tolerance capacity. P. euphratica suspended cells heat‐shocked for 20 min were able to tolerate temperatures up to 45°C. Heat‐associated events as PCD and ROS production were suggested not to be implicated in the occurring cell death. Supported by the use of publicly available A. thaliana expression data and other webbased tools and resources, a reverse genetics strategy was followed for the identification of novel determinants for heat stress tolerance (HZF and HRR). In silico analysis revealed that both genes putatively encode effector proteins involved in different stages of the heat stress response. Moreover, HZF and HRR were found to be co‐regulated with genes already implicated in the regulation of heat responses. Functional characterization of HZF was primarily supported by the use of several web‐based tools and resources specifically created for Arabidopsis functional analysis. HZF was found to be a zinc finger family protein containing a conserved C3H2C3‐type RING domain and its possible role as E3 ubiquitin ligase was suggested. To pursue with reverse genetics approaches for identifying heat stress‐associated mutations, a phenotypic analysis based on germination and seedling survival assays was proposed. Temperatures and periods of treatment were diversely combined to test basal thermotolerance in either seeds or 7‐day‐old seedlings, or acquired thermotolerance only in 7‐ day‐old seedlings. The effectiveness of the proposed protocols was illustrated by detection of heat‐associated phenotypes in two mutants (hot1‐3 and atrbohD) previously identified to be thermotolerance defective. Regarding germination assays, special attention should be given to the time‐course evaluation of the number of germinated seeds for an accurate phenotypic detection. A delayed germination was observed in hzf mutant seeds in the following days after heat treatment when compared to wild‐type seeds, suggesting a role for HZF in the transition from dormant to germinating state. HZF was then suggested to mediate the ubiquitination of a regulator protein implicated in promoting seed dormancy or repressing germination upon heat stress. This function seems to be mainly assured by a redundant gene product (L‐HZF) under standard conditions, since similar germination timing was observed for hzf and wild‐type seeds. Maximum HZF transcript accumulation in heat‐treated (38°C for 1 h) wild‐type seedlings was achieved 15 min after heat treatment, suggesting also the HZF involvement in the initial phase of heat stress response. Expression vectors suitable for overexpression studies and in situ analysis were constructed and used to transform wild‐type Arabidopsis plants. The transgenic T3 plants will be soon available for further experiments that will contribute to elucidate the specific role of HZF in thermotolerance. The complete functional characterization of HZF, currently in progress, will provide novel information that would contribute to the dissection of its particular role in plant thermotolerance.

Mots-clés : Arabidopsis thaliana, Functional genomics, Heat stress responsive genes, Populus euphratica, Reverse genetics,Thermotolerance, Genes de resposta ao stress térmico, Genética inversa, Genómica funcional, Populus euphratica, Termotolerância

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