Estudi de l'especialització funcional dels isoenzims citosòlics de la farnesildifosfat sintasa d'Arabidopsis thaliana

[cat] Les farnesildifosfat sintases (FPS) catalitzen la condensació de dues molècules d'isopentenildifosfat (IPP) amb una molècula de dimetilal.lildifosfat (DMAPP) per produir farnesildifosfat (FPP; C15). Tant els substrats com el producte de la reacció catalitzada per les FPS ocupen una posició molt important en la via del mevalonat de síntesi d'isoprenoides, ja que són punt de partida de nombroses ramificacions cap a la síntesi de diversos productes finals. A Arabidopsis thaliana existeix una petita família multigènica integrada pels gens FPS1 i FPS2 , que codifiquen tres isoenzims FPS: FPS1S, FPS1L i FPS2. L'isoenzim FPS1L conté un pèptid de trànsit que li confereix una localització mitocondrial, mentre que FPS1S i previsiblement FPS2, tenen una localització citosòlica. A l'inici d'aquest treball, s'havien clonat els gens FPS1 i FPS2 , i s'havien definit els seus patrons d'expressió espaial i temporal mitjançant l'anàlisi de plantes transgèniques portadores de gens quimèrics formats pels promotors d'ambdós gens fusionats al gen reporter uidA d' E. Coli . També s'havia estudiat el paper dels isoenzims FPS1S i FPS1L en la via del mevalonat a través de la caracterització de plantes transgèniques que sobreexpressaven aquests isoenzims. La localització de l'isoenzim FPS1L a les mitocòndries ja és, en sí mateixa, una evidència d'especialització funcional. En el cas dels isoenzims FPS1S i FPS2, el fet de pensar en l'existència d'una especialització funcional deriva dels seus patrons d'expressió diferencials. Amb l'objectiu d'analitzar el paper de l'isoenzim FPS2 en la via del mevalonat, i obtenir evidències de l'especialització funcional dels isoenzims FPS1S i FPS2, en aquesta tesi es duen a terme diferents abordatges experimentals basats en l'obtenció i caracterització de mutants d' A. Thaliana amb guany i pèrdua de funció dels gens FPS1 i FPS2 . Per una banda, mitjançant l'estudi dels efectes de la sobreexpressió de l'isoenzim FPS2 i de la sobreexpressió simultània dels isoenzims FPS1S i FPS2. D'altra banda, a través de l'anàlisi de mutants amb pèrdua de funció dels gens FPS1 i FPS2 i del doble mutant fps1:fps2 . Aquests abordatges es complementen amb l'estudi dels efectes de la sobreexpressió en A. Thaliana de proteïnes quimèriques FPS1S/FPS2 ( domain swapping ), l'elaboració de models estructurals tridimensionals dels isoenzims FPS1S i FPS2 i l'anàlisi dels perfils d'expressió gènica en mutants amb pèrdua de funció dels isoenzims FPS. En aquest treball es demostra que els isoenzims FPS1S i FPS2, tot i catalitzar la síntesi del mateix producte, tenir la mateixa localització subcel.lular i uns paràmetres cinètics similars, produeixen efectes molt diferents quan són sobreexpressats en A. thaliana. La presència d'un únic isoenzim FPS és suficient per garantir la viabilitat de les plantes, ara bé, la pèrdua de funció de l'isoenzim FPS2 desencadena una resposta metabòlica compensatòria a les llavors. En conjunt, tots els resultats indiquen que els isoenzims citosòlics FPS1S i FPS2 desenvolupen funcions majoritàriament redundants, tot i que presenten una certa especialització funcional que sembla derivar del patró d'expressió especialitzat de cadascun dels gens FPS .[eng] Farnesyldiphosphate synthases (FPS) catalyze the condensation of two molecules of isopentenyldiphosphate (IPP) with its isomer dimethylallyldiphosphate (DMAPP) to produce farnesyldiphosphate (FPP; C15). FPS is located at a key position in the mevalonic acid pathway (MVA), as both the substrates and the reaction product are precursors of a variety of isoprenoid end-products. Arabidopsis thaliana contains a small FPS gene family consisting of two genes, FPS1 and FPS2 , encoding three FPS isoforms: FPS1S, FPS1L and FPS2. FPS1L isoform is located in the mitochondria, whereas FPS1S and likely FPS2, are both localized in the cytosol. The differential patterns of expression of FPS1 and FPS2 , strongly suggest a functional specialization of the encoded FPS1S and FPS2 isoforms. The aim of this thesis is to analyze the role of FPS2 isozyme in the MVA pathway and to obtain evidence of a functional specialization of FPS1S and FPS2 isozymes. Different experimental approaches have been undertaken using A. thaliana mutants with gain and loss of function of FPS1S and FPS2. We have investigated the effects of FPS2 overexpression and the effects of the overexpression of both FPS isozymes simultaneously. We have also characterized A. thaliana knock-out mutants in both FPS genes and the double knock-out mutant fps1:fps2 . Analysis of the effects caused by the overexpression of chimeric FPS1S/FPS2 proteins in A. thaliana , 3D modelling of FPS isozymes, and microarray analysis of FPS knock-out mutants are also included in this study. Results demonstrate that, although FPS1S and FPS2 isozymes synthesize the same reaction product, have the same subcellular localization and have similar kinetic parameters, its overexpression produce different effects in A. thaliana . The existence of a single FPS isozyme is enough to assure plant viability. However, FPS2 loss of function triggers a compensatory metabolic response in A. thaliana seeds. All these results indicate that FPS1S and FPS2 play redundant functions in the MVA pathway, although they also show a certain level of functional specialization

Multilevel control of arabidopsis 3-hydroxy-3-methylglutaryl coenzyme A reductase by protein phosphatase 2A

Plants synthesize a myriad of isoprenoid products that are required both for essential constitutive processes and for adaptive responses to the environment. The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes a key regulatory step of the mevalonate pathway for isoprenoid biosynthesis and is modulated by many endogenous and external stimuli. In spite of that, no protein factor interacting with and regulating plant HMGR in vivo has been described so far. Here, we report the identification of two B99 regulatory subunits of protein phosphatase 2A (PP2A), designated B99a and B99b, that interact with HMGR1S and HMGR1L, the major isoforms of Arabidopsis thaliana HMGR. B99a and B99b are Ca2+ binding proteins of the EF-hand type. We show that HMGR transcript, protein, and activity levels are modulated by PP2A in Arabidopsis. When seedlings are transferred to salt-containing medium, B99a and PP2A mediate the decrease and subsequent increase of HMGR activity, which results from a steady rise of HMGR1-encoding transcript levels and an initial sharper reduction of HMGR protein level. In unchallenged plants, PP2A is a posttranslational negative regulator of HMGR activity with the participation of B99b. Our data indicate that PP2A exerts multilevel control on HMGR through the fivemember B99 protein family during normal development and in response to a variety of stress conditions

Multilevel control of arabidopsis 3-hydroxy-3-methylglutaryl coenzyme A reductase by protein phosphatase 2A

Plants synthesize a myriad of isoprenoid products that are required both for essential constitutive processes and for adaptive responses to the environment. The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes a key regulatory step of the mevalonate pathway for isoprenoid biosynthesis and is modulated by many endogenous and external stimuli. In spite of that, no protein factor interacting with and regulating plant HMGR in vivo has been described so far. Here, we report the identification of two B99 regulatory subunits of protein phosphatase 2A (PP2A), designated B99a and B99b, that interact with HMGR1S and HMGR1L, the major isoforms of Arabidopsis thaliana HMGR. B99a and B99b are Ca2+ binding proteins of the EF-hand type. We show that HMGR transcript, protein, and activity levels are modulated by PP2A in Arabidopsis. When seedlings are transferred to salt-containing medium, B99a and PP2A mediate the decrease and subsequent increase of HMGR activity, which results from a steady rise of HMGR1-encoding transcript levels and an initial sharper reduction of HMGR protein level. In unchallenged plants, PP2A is a posttranslational negative regulator of HMGR activity with the participation of B99b. Our data indicate that PP2A exerts multilevel control on HMGR through the fivemember B99 protein family during normal development and in response to a variety of stress conditions

Characterization of Arabidopsis FPS isozymes and FPS gene expression analysis provide insight into the biosynthesis of isoprenoid precursors in seeds

Arabidopsis thaliana contains two genes encoding farnesyl diphosphate (FPP) synthase (FPS), the prenyl diphoshate synthase that catalyzes the synthesis of FPP from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In this study, we provide evidence that the two Arabidopsis short FPS isozymes FPS1S and FPS2 localize to the cytosol. Both enzymes were expressed in E. coli, purified and biochemically characterized. Despite FPS1S and FPS2 share more than 90% amino acid sequence identity, FPS2 was found to be more efficient as a catalyst, more sensitive to the inhibitory effect of NaCl, and more resistant to thermal inactivation than FPS1S. Homology modelling for FPS1S and FPS2 and analysis of the amino acid differences between the two enzymes revealed an increase in surface polarity and a greater capacity to form surface salt bridges of FPS2 compared to FPS1S. These factors most likely account for the enhanced thermostability of FPS2. Expression analysis of FPS::GUS genes in seeds showed that FPS1 and FPS2 display complementary patterns of expression particularly at late stages of seed development, which suggests that Arabidopsis seeds have two spatially segregated sources of FPP. Functional complementation studies of the Arabidopsis fps2 knockout mutant seed phenotypes demonstrated that under normal conditions FPS1S and FPS2 are functionally interchangeable. A putative role for FPS2 in maintaining seed germination capacity under adverse environmental conditions is discussed