Unveiling the biological role of stigmasterol biosynthesis in tomato plants

Summary In plants, the sterol biosynthetic pathway leads to the production of a complex mixture of end-products, among which β-sitosterol, stigmasterol, campesterol, and cholesterol are the most abundant ones. Stigmasterol, the end-product of the 24-ethyl sterol branch is synthesized from β-sitosterol by the action of sterol C22-desaturase (C22DES). Despite C22DES was identified about a decade ago, there is still a lack of knowledge about several relevant aspects related to the structure and function of this enzyme. Furthermore, several studies have reported the occurrence of changes in the stigmasterol to β-sitosterol ratio (STIG/SIT) during plant development and in their responses to environmental stimuli which have been related with the activity of C22DES. However, the biological relevance of the changes in stigmasterol levels is currently unknown. Based on this, the main objective of present work has been the elucidation of the role of stigmasterol during plant growth and development as well as in its response to environmental challenges. Tomato (Solanum lycopersicum cv. MicroTom) was selected for this studies taking advantage that is one of the few plants in which C22DES is encoded by a single-copy gene. The goals of this thesis have been: (1) the functional and structural characterization of C22DES in terms of subcellular localization and mechanism of action, and (2) the evaluation of the role of stigmasterol biosynthesis in tomato plant growth, development and in responses to biotic and abiotic stresses. The results obtained in this thesis have confirmed that C22DES is targeted and retained in the endoplasmic reticulum (ER)-membrane. C22DES consists of two well-differentiated domains: a single N-terminal transmembrane-helix domain (TMH1), which is sufficient for the ER-membrane targeting and retention, and a globular domain that also contacts with the ER-membrane. The globular domain may also interact and be retained in the ER in the absence of TMH1, but it is enzymatically inactive, revealing the requirement of the N-terminal membrane domain for enzyme activity. The in silico analysis of the TMH1 region revealed several features that could be involved in substrate recognition and binding. Transgenic tomato plants with altered STIG/SIT ratios have been generated by overexpression, amiRNA-mediated silencing, and knock-out of the C22DES gene. The obtained results have shown that lack of stigmasterol is not lethal for the plant since the C22des- knock-out plants are able to complete their life cycle. However, C22des- knock-out mutant showed several phenotypic abnormalities related to growth and developmental processes. The C22des- knock-out plants also show increased susceptibility to Botrytis cinerea infection. The data provided in this thesis contribute to expand the current knowledge on the mechanism of action of stigmasterol and C22DES in tomato plants and set the basis for further studies focused on unraveling the mechanisms involved in the regulation of C22DES activity. The C22des- knock-out mutant generated in this work also provides a unique and highly relevant tool to evaluate the role of stigmasterol in plants.