Role of conjugated sterols homeostasis in plant development and stress response
[eng] Phytosterols are a family of triterpenoid compounds that occur in free form (FS) or conjugated as steryl esters (SE), steryl glycosides (SG) and acylated steryl glycosides (ASG). Free sterols and their glycosylated derivatives are structural components of the cell membranes that play a key rol...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2024 |
| País: | España |
| Institución: | Universidad de Barcelona |
| Repositorio: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/220507 |
| Acceso en línea: | https://hdl.handle.net/2445/220507 http://hdl.handle.net/10803/694273 |
| Access Level: | acceso abierto |
| Palabra clave: | Genòmica Tomàquets Fitosterols Glucòsids Esterificació Desenvolupament de les plantes Genomics Tomatoes Phytosterols Glucosides Esterification Plant development |
| Sumario: | [eng] Phytosterols are a family of triterpenoid compounds that occur in free form (FS) or conjugated as steryl esters (SE), steryl glycosides (SG) and acylated steryl glycosides (ASG). Free sterols and their glycosylated derivatives are structural components of the cell membranes that play a key role in maintaining their fluidity and permeability, while SE are stored in cytoplasmic lipid droplets, where they serve as a reservoir of sterols that can be accumulated or mobilized depending on the cellular needs. In SG, the C3 hydroxyl group of the sterol backbone is linked through a glycosydic bond to a sugar moiety, which may have a long-chain fatty acid esterified to the hydroxyl group at position C6 leading to ASG. The hydroxyl group of FS can also be esterified to a long-chain fatty acid producing SE. FS are the common precursors of SG and SE, which are synthesized by the UDP-glucose:sterol glycosyltransferases (SGT) and the acyl-CoA/phospholipid:sterol acyltransferases (ASAT/PSAT), respectively . These enzymes have been cloned and characterized in different plants, including tomato, where our group has characterized SlASAT1, SlPSAT1 and the four SGTs (SlSGT1-SlSGT4). However, the specific role of these enzymes in the conjugated sterol metabolism is still not well understood. To investigate the biological function of conjugated sterols, in this thesis we have used tomato CRISPR/Cas9 slpsat1, slasat1 and slpsat1xslasat1 knock-out mutants, and SlSGT3 overexpressing plants (SlSGT3-ox) to investigate how changes in the profile of conjugated sterols may affect tomato growth and development and its response to biotic stress. The characterization of slpsat1 and slasat1 single and double knock-out mutants revealed that SlPSAT1 has a predominant role in tomato SE biosynthesis while SlASAT1 would mainly regulate the flux of the sterol pathway. The strong reduction of SE levels in the leaves, fruits and seeds associated to PSAT1 and ASAT1 inactivation affect in different ways several important cellular and physiological processes, leading to reduced leaf number of lipid droplets, enhanced seed germination speed, early leaf senescence, reduction of the overall plant size, and increased resistance of fruits to Botrytis cinerea infection. Analysis of the overall profile of free and conjugated sterols in fruits and seeds of the slpsat31 x slasat34 mutant revealed also different qualitative and quantitative effects on the composition of free and glycosylated sterols. While in fruits impaired SE biosynthesis resulted in higher levels of FS and SG and lower levels of ASG compared to the WT, no changes were observed in seed FS levels while those of SG and ASG increased drastically. Moreover, the impact of the changes in sterol composition on the metabolome of fruit and seeds has been studied, and the results have shown a differential impact on fruit and seed metabolomes, although in both cases disruption of the free and conjugated sterol balance in the slpsat31 x slasat34 mutant induces autophagy in both organs. SlSGT3-ox plants show a significant increase in SG levels, accompanied by a marked reduction in SE in both leaves and fruits. This resulted in a strong vegetative phenotype characterized by a clear dwarfism and thickening of the internodes, while fruits are firmer than WT fruits and show a marked delay in ripening, which is most likely due to a delay in the peak of ethylene production. These results, along with the triple response to ethylene observed in SlSGT3-ox seedlings, suggests a relationship between SG and the synthesis and/or perception-sensitivity to this hormone. To gain some insight about the molecular mechanisms underlying these physiological effects, we performed a transcriptomic analysis of SlSGT3-ox seedlings. The results showed a misregulation, among others, of genes involved in developmental processes, cell wall biogenesis, cytoskeleton and organelle organization, detoxification and responses to different stimuli, that might help to explain some of the observed phenotypes. To obtain fruits equivalent to those in SlSGT3-ox plants, but avoiding the observed alterations in plant development, plants overexpressing SlSGT3 under E8 fruit specific ethylene responsive promoter have been generated and preliminary characterized. Finally, a method for isolating lipid rafts from leaves and roots has been set up. This will allow the study of the effects of altered levels of sterols on the lipid and protein composition of these plasma membrane subdomains, which have been reported to be involved in a variety of plant cell biological processes. Too gain further insight on the intracellular localization of sterols some preliminary analysis of the spatial distribution of sterols have been carried out using MALDI-MSI analysis. Altogether, the results obtained in this work demonstrate that conjugated sterols play an important role in the growth and development of tomato plants and fruits, as well as in the plant response to biotic stress. The results of this thesis project have set the basis for further studies aimed at deciphering the molecular mechanisms underlaying the perturbations observed in these physiological processes. |
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