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La autofagia es un proceso catabólico ampliamente conservado en eucariotas por el cual se degradan componentes celulares incluyendo proteínas, ribosomas u orgánulos completos para mantener la homeostasis celular. La síntesis de ácidos grasos se localiza en el estroma del cloroplasto de plantas y algas. Los ácidos grasos sintetizados de novo por la enzima ácido graso sintasa (FAS) se utilizan como precursores de los lípidos esenciales para la estructura del cloroplasto y su función. El objetivo principal de esta tesis es investigar la conexión entre el proceso de autofagia y la inhibición de la síntesis de ácidos grasos en el cloroplasto de la microalga modelo Chlamydomonas reinhardtii. Los resultados de esta tesis demuestran que la inhibición de la enzima FAS mediante la droga cerulenina, un inhibidor específico de esta enzima, activa el proceso de autofagia. El tratamiento con esta droga disminuye la abundancia del lípido monogalactosil diacilglicerol (MGDG), el lípido mayoritario de las membranas plastídicas. Además, este tratamiento disminuye la eficiencia fotosintética y aumenta los niveles de especies reactivas del oxígeno (ROS). Igualmente, mediante microscopía electrónica de transmisión se observó que las membranas tilacoidales presentan un alto grado de empaquetamiento y un mayor número de membranas por apilamiento en células tratadas con cerulenina. Por otro lado, el análisis transcriptómico realizado en estas células mostró la inducción de genes de respuesta a estrés en el cloroplasto y la acumulación de transcritos relacionados con la detoxificación de ROS. Así, nuestros resultados indican que existe una relación entre el metabolismo de lípidos, la respuesta a estrés en el cloroplasto y el proceso de autofagia en Chlamydomonas reinhardtii. Para profundizar en el estudio de la inhibición de la enzima FAS y su conexión con la autofagia en este organismo modelo, se generó un mutante deficiente de autofagia mediante el sistema de edición genético CRISPR/Cas9.

Microalgae constitute a highly diverse group of photosynthetic microorganisms that are widely distributed on Earth. The rich diversity of microalgae arose from endosymbiotic events that took place early in the evolution of eukaryotes and gave rise to multiple lineages including green algae, the ancestors of land plants. In addition to their fundamental role as the primary source of marine and freshwater food chains, microalgae are essential producers of oxygen in the planet and a major biotechnological target for sustainable biofuel production and CO2 mitigation. Microalgae integrate light and nutrient signals to regulate cell growth. Recent studies identified the target of rapamycin (TOR) kinase as central regulator of cell growth and nutrient sensor in microalgae. TOR promotes protein synthesis and regulates processes that are induced under nutrient stress such as autophagy and the accumulation of triacylglycerol and starch. A detailed analysis of representative genomes from the entire microalgal lineage revealed the high conservation of central components of the TOR pathway likely present in the last eukaryotic common ancestor and the loss of specific TOR signaling elements at an early stage in the evolution of microalgae. Here we examine the evolutionary conservation of TOR signaling components in diverse microalgae and discuss recent progress on the study of this signaling pathway in these organisms.

Stress and nutrient availability influence cell proliferation through complex intracellular signalling networks. In a previous study it was found that pyro-inositol polyphosphates (InsP7 and InsP8) produced by VIP1 kinase, and target of rapamycin (TOR) kinase signalling interacted synergistically to control cell growth and lipid metabolism in the green alga Chlamydomonas reinhardtii. However, the relationship between InsPs and TOR was not completely elucidated. We used an in vivo assay for TOR activity together with global proteomic and phosphoproteomic analyses to assess differences between wild-type and vip1-1 in the presence and absence of rapamycin. We found that TOR signalling is more severely affected by the inhibitor rapamycin in a vip1-1 mutant compared with wild-type, indicating that InsP7 and InsP8 produced by VIP1 act independently but also coordinately with TOR. Additionally, among hundreds of differentially phosphorylated peptides detected, an enrichment for photosynthesis-related proteins was observed, particularly photosystem II proteins. The significance of these results was underscored by the finding that vip1-1 strains show multiple defects in photosynthetic physiology that were exacerbated under high light conditions. These results suggest a novel role for inositol pyrophosphates and TOR signalling in coordinating photosystem phosphorylation patterns in Chlamydomonas cells in response to light stress and possibly other stresses.

Target of rapamycin complex 1 (TORC1) is a central regulator of cell growth. It balances anabolic and catabolic processes in response to nutrients, growth factors, and energy availability. Nitrogen- and carbon-containing metabolites have been shown to activate TORC1 in yeast, animals, and plants. Here, we show that phosphorus (P) regulates TORC1 signaling in the model green alga Chlamydomonas (Chlamydomonas reinhardtii) via LST8, a conserved TORC1 subunit that interacts with the kinase domain of TOR. P starvation results in a sharp decrease in LST8 abundance and downregulation of TORC1 activity. A hypomorphic lst8 mutation resulted in decreased LST8 abundance, and it both reduced TORC1 signaling and altered the cellular response to P starvation. Additionally, we found that LST8 levels and TORC1 activity were not properly regulated in a mutant defective in the transcription factor PSR1, which is the major mediator of P deprivation responses in Chlamydomonas. Unlike wild-type cells, the psr1 mutant failed to downregulate LST8 abundance and TORC1 activity when under P limitation. These results identify PSR1 as an upstream regulator of TORC1 and demonstrate that TORC1 is a key component in P signaling in Chlamydomonas.

Hydrogen sulfide is a signaling molecule that regulates essential processes in plants, such as autophagy. In Arabidopsis (Arabidopsis thaliana), hydrogen sulfide negatively regulates autophagy independently of reactive oxygen species via an unknown mechanism. Comparative and quantitative proteomic analysis was used to detect abscisic acid-triggered persulfidation that reveals a main role in the control of autophagy mediated by the autophagy-related (ATG) Cys protease AtATG4a. This protease undergoes specific persulfidation of Cys170 that is a part of the characteristic catalytic Cys-His-Asp triad of Cys proteases. Regulation of the ATG4 activity by persulfidation was tested in a heterologous assay using the Chlamydomonas reinhardtii CrATG8 protein as a substrate. Sulfide significantly and reversibly inactivates AtATG4a. The biological significance of the reversible inhibition of the ATG4 by sulfide is supported by the results obtained in Arabidopsis leaves under basal and autophagy-activating conditions. A significant increase in the overall ATG4 proteolytic activity in Arabidopsis was detected under nitrogen starvation and osmotic stress and can be inhibited by sulfide. Therefore, the data strongly suggest that the negative regulation of autophagy by sulfide is mediated by specific persulfidation of the ATG4 protease.

Target of rapamycin (TOR) kinase is a conserved regulator of cell growth whose activity is modulated in response to nutrients, energy and stress. Key proteins involved in the pathway are conserved in the model photosynthetic microalga Chlamydomonas reinhardtii, but the substrates of TOR kinase and downstream signaling network have not been elucidated. Our study provides a new resource for investigating the phosphorylation networks governed by the TOR kinase pathway in Chlamydomonas. We used quantitative phosphoproteomics to investigate the effects of inhibiting Chlamydomonas TOR kinase on dynamic protein phosphorylation. Wild-type and AZD-insensitive Chlamydomonas strains were treated with TOR-specific chemical inhibitors (rapamycin, AZD8055 and Torin1), after which differentially affected phosphosites were identified. Our quantitative phosphoproteomic dataset comprised 2547 unique phosphosites from 1432 different proteins. Inhibition of TOR kinase caused significant quantitative changes in phosphorylation at 258 phosphosites, from 219 unique phosphopeptides. Our results include Chlamydomonas homologs of TOR signaling-related proteins, including a site on RPS6 with a decrease in phosphorylation. Additionally, phosphosites on proteins involved in translation and carotenoid biosynthesis were identified. Follow-up experiments guided by these phosphoproteomic findings in lycopene beta/epsilon cyclase showed that carotenoid levels are affected by TORC1 inhibition and carotenoid production is under TOR control in algae.

Autophagy is an intracellular catabolic process that allows cells to recycle unneeded or damaged material to maintain cellular homeostasis. This highly dynamic process is characterized by the formation of double-membrane vesicles called autophagosomes, which engulf and deliver the cargo to the vacuole. Flow of material through the autophagy pathway and its degradation in the vacuole is known as autophagic flux, and reflects the autophagic degradation activity. A number of assays have been developed to determine autophagic flux in yeasts, mammals, and plants, but it has not been examined yet in algae. Here we analyzed autophagic flux in the model green alga Chlamydomonas reinhardtii. By monitoring specific autophagy markers such as ATG8 lipidation and using immunofluorescence and electron microscopy techniques, we show that concanamycin A, a vacuolar ATPase inhibitor, blocks autophagic flux in Chlamydomonas. Our results revealed that vacuolar lytic function is needed for the synthesis of triacylglycerols and the formation of lipid bodies in nitrogen- or phosphate-starved cells. Moreover, we found that concanamycin A treatment prevented the degradation of ribosomal proteins RPS6 and RPL37 under nitrogen or phosphate deprivation. These results indicate that autophagy might play an important role in the regulation of lipid metabolism and the recycling of ribosomal proteins under nutrient limitation in Chlamydomonas

Algae undergo a complete metabolic transformation under stress by arresting cell growth, inducing autophagy and hyperaccumulating biofuel precursors such as triacylglycerols and starch. However, the regulatory mechanisms behind this stress-induced transformation are still unclear. Here, we use biochemical, mutational, and “omics” approaches to demonstrate that PI3K signaling mediates the homeostasis of energy molecules and influences carbon metabolism in algae. In Chlamydomonas reinhardtii, the inhibition and knockdown (KD) of algal class III PI3K led to significantly decreased cell growth, altered cell morphology, and higher lipid and starch contents. Lipid profiling of wild-type and PI3K KD lines showed significantly reduced membrane lipid breakdown under nitrogen starvation (-N) in the KD. RNA-seq and network analyses showed that under -N conditions, the KD line carried out lipogenesis rather than lipid hydrolysis by initiating de novo fatty acid biosynthesis, which was supported by tricarboxylic acid cycle down-regulation and via acetyl-CoA synthesis from glycolysis. Remarkably, autophagic responses did not have primacy over inositide signaling in algae, unlike in mammals and vascular plants. The mutant displayed a fundamental shift in intracellular energy flux, analogous to that in tumor cells. The high free fatty acid levels and reduced mitochondrial ATP generation led to decreased cell viability. These results indicate that the PI3K signal transduction pathway is the metabolic gatekeeper restraining biofuel yields, thus maintaining fitness and viability under stress in algae. This study demonstrates the existence of homeostasis between starch and lipid synthesis controlled by lipid signaling in algae and expands our understanding of such processes, with biotechnological and evolutionary implications.

Fatty acids are synthesized in the stroma of plant and algal chloroplasts by the fatty acid synthase complex. Newly synthesized fatty acids are then used to generate plastidial lipids that are essential for chloroplast structure and function. Here, we show that inhibition of fatty acid synthesis in the model alga Chlamydomonas reinhardtii activates autophagy, a highly conserved catabolic process by which cells degrade intracellular material under adverse conditions to maintain cell homeostasis. Treatment of Chlamydomonas cells with cerulenin, a specific fatty acid synthase inhibitor, stimulated lipidation of the autophagosome protein ATG8 and enhanced autophagic flux. We found that inhibition of fatty acid synthesis decreased monogalactosyldiacylglycerol abundance, increased lutein content, down-regulated photosynthesis, and increased the production of reactive oxygen species. Electron microscopy revealed a high degree of thylakoid membrane stacking in cerulenin-treated cells. Moreover, global transcriptomic analysis of these cells showed an up-regulation of genes encoding chloroplast proteins involved in protein folding and oxidative stress and the induction of major catabolic processes, including autophagy and proteasome pathways. Thus, our results uncovered a link between lipid metabolism, chloroplast integrity, and autophagy through a mechanism that involves the activation of a chloroplast quality control system.

Microspores are reprogrammed towards embryogenesis by stress. Many microspores die after this stress, limiting the efficiency of microspore embryogenesis. Autophagy is a degradation pathway that plays critical roles in stress response and cell death. In animals, cathepsins have an integral role in autophagy by degrading autophagic material; less is known in plants. Plant cathepsins are papain-like C1A cysteine proteases involved in many physiological processes, including programmed cell death. We have analysed the involvement of autophagy in cell death, in relation to cathepsin activation, during stress-induced microspore embryogenesis in Hordeum vulgare. After stress, reactive oxygen species (ROS) and cell death increased and autophagy was activated, including HvATG5 and HvATG6 up-regulation and increase of ATG5, ATG8, and autophagosomes. Concomitantly, cathepsin L/F-, B-, and H-like activities were induced, cathepsin-like genes HvPap-1 and HvPap-6 were up-regulated, and HvPap-1, HvPap-6, and HvPap-19 proteins increased and localized in the cytoplasm, resembling autophagy structures. Inhibitors of autophagy and cysteine proteases reduced cell death and promoted embryogenesis. The findings reveal a role for autophagy in stress-induced cell death during microspore embryogenesis, and the participation of cathepsins. Similar patterns of activation, expression, and localization suggest a possible connection between cathepsins and autophagy. The results open up new possibilities to enhance microspore embryogenesis efficiency with autophagy and/or cysteine protease modulators.

Autophagy is an intracellular catabolic system that delivers cytoplasmic constituents and organelles in the vacuole. This degradative process is mediated by a group of proteins coded by autophagy-related (ATG) genes that are widely conserved from yeasts to plants and mammals. Homologs of ATG genes have been also identified in algal genomes including the unicellular model green alga Chlamydomonas reinhardtii. The development of specific tools to monitor autophagy in Chlamydomonas has expanded our current knowledge about the regulation and function of this process in algae. Recent findings indicated that autophagy is regulated by redox signals and the TOR network in Chlamydomonas and revealed that this process may play in important role in the control of lipid metabolism and ribosomal protein turnover in this alga. Here, we will describe the different techniques and approaches that have been reported to study autophagy and autophagic flux in Chlamydomonas

Autophagy is a major catabolic pathway by which eukaryotic cells deliver unnecessary or damaged cytoplasmic material to the vacuole for its degradation and recycling in order to maintain cellular homeostasis. Control of autophagy has been associated with the production of reactive oxygen species in several organisms, including plants and algae, but the precise regulatory molecular mechanisms remain unclear. Here, we show that the ATG4 protease, an essential protein for autophagosome biogenesis, plays a central role for the redox regulation of autophagy in the model green alga Chlamydomonas reinhardtii. Our results indicate that the activity of C. reinhardtii ATG4 is regulated by the formation of a single disulfide bond with a low redox potential that can be efficiently reduced by the NADPH/thioredoxin system. Moreover, we found that treatment of C. reinhardtii cells with norflurazon, an inhibitor of carotenoid biosynthesis that generates reactive oxygen species and triggers autophagy in this alga, promotes the oxidation and aggregation of ATG4. We propose that the activity of the ATG4 protease is finely regulated by the intracellular redox state, and it is inhibited under stress conditions to ensure lipidation of ATG8 and thus autophagy progression in C. reinhardtii.

[EN] Autophagy is a conserved catabolic process in eukaryotic organisms by which cells degrade or recycle internal constituents under stress or optimal conditions to produce new macromolecules or energy. Chamydomonas reinhardtii, in which autophagy process is conserved, is a unicellular green alga commonly used as a model organism for photosynthesis research. Recently, its importance has grown up commercially and biotechnologically because of its application in biofuel production, as this organism is able to synthetize triacylglycerols (TAGs). Due to its high potential, a number of recent studies have been reported to understand the parameters involved in lipids synthesis. In this context, it is known that the starch-deficient mutant strain, sta6, accumulates more lipids than wild type cells. Furthermore, lipid levels increase when cells are grown under nitrogen deficiency. The lack of nitrogen generates stress which in turn triggers autophagy. The relationship between autophagy, starch and lipid metabolism is analyzed in this work. Here, we report that starch deficiency in cells growing with or without nitrogen does not induce autophagy. However, when cells were treated with cerulenin, a fatty acids synthase inhibitor, autophagy is induced because of fatty acids profile modification and especially because of the decrease of the glucolipid MGDG, which is part of thylakoid membranes and photosystems. The relationship between autophagy and MGDG fall has never been described before., [ES] La autofagia es un proceso catabólico de degradación y reciclado de componentes celulares internos conservado en eucariotas que se produce ante un estrés o condiciones óptimas de crecimiento para generar nuevas macromoléculas o energía. Chlamydomonas reinhardtii es una microalga verde unicelular muy utilizada en el laboratorio como organismo modelo para estudios fotosintéticos y en la que está conservado este proceso. Recientemente, ha cobrado interés a nivel biotecnológico y comercial debido a su aplicación en biocombustibles, ya que son capaces de producir Triacilgliceroles (TAGs). Desde que se demostró el gran potencial de estas microalgas, se han realizado diversos estudios para concretar los parámetros que afectan a su síntesis. En este contexto, se conoce que la estirpe mutante en la síntesis de almidón, sta6, acumula más lípidos que la estirpe silvestre y que este contenido aumenta en medio en deficiencia de nitrógeno, condición de estrés que activa la autofagia. En este trabajo se ha investigado la relación de la autofagia con la producción de almidón y la acumulación de lípidos. Nuestros resultados indican que la ausencia de almidón en medio con y sin nitrógeno no induce la autofagia. Sin embargo, al tratar con cerulenina, compuesto inhibidor de la ácido graso sintasa, la autofagia se induce debido a la modificación del perfil de ácido grasos de la célula y, sobre todo, debido a la reducción del glucolípido MGDG, lípido presente en la membrana tilacoidal y en los fotosistemas. Esta relación entre la inducción de la autofagia y la disminución en los niveles de MGDG no ha sido descrita hasta el momento en ningún sistema.

The accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) results in the activation of stress responses such as the unfolded protein response or the catabolic process of autophagy to ultimately recover cellular homeostasis. ER stress also promotes the production of reactive oxygen species (ROS), which play an important role in autophagy regulation. However, it remains unknown whether ROS are involved in ER stress-induced autophagy. In the present study, we provide evidence connecting redox imbalance caused by ER stress and autophagy activation in the model unicellular green alga Chlamydomonas reinhardtii. Treatment of Chlamydomonas cells with the ER stressors tunicamycin or dithiothreitol resulted in upregulation of the expression of genes encoding ER resident ERO1 oxidoreductase and protein disulfide isomerases. ER stress also triggered autophagy in Chlamydomonas based on the protein abundance, lipidation, cellular distribution and mRNA levels of the autophagy marker ATG8. Moreover, an increase in the oxidation of the glutathione pool and in the expression of oxidative stress-related genes were detected in tunicamycin-treated cells. Our results revealed that the antioxidant glutathione partially suppressed ER stress-induced autophagy and decreased the toxicity of tunicamycin, suggesting that oxidative stress participates in the control of autophagy in response to ER stress in Chlamydomonas. In close agreement, we also found that autophagy activation by tunicamycin was more pronounced in the Chlamydomonas sor1 mutant, which shows increased expression of oxidative stress-related genes

Hydrogen sulfide is already recognized as an important signaling molecule in mammalian systems, and emerging data suggest that H2S is a signaling molecule just as important as nitric oxide (NO) and H2O2 in plants. Although sulfide is generated in chloroplasts and mitochondria, it is present predominantly in the charged HS- form due to the basic pH inside both organelles, thus requiring an active transporter, which is yet to be identified, to be released. In Arabidopsis, we found that the cytosolic l-cysteine desulfhydrase DES1 is involved in the degradation of cysteine, and therefore responsible for the generation of H2S in this cellular compartment. DES1 deficiency leads to the induction of autophagy. Moreover, we have demonstrated that sulfide in particular exerts a general effect on autophagy through negative regulation, in a way unrelated to nutrient deficiency. The mechanisms of H2S action and its molecular targets are largely unknown, although in animal systems, protein S-sulfhydration has been proposed as a mechanism for sulfide-mediated signaling.

In Arabidopsis thaliana, DES1 is the only identified l-Cysteine desulfhydrase located in the cytosol, and it is involved in the degradation of cysteine and the concomitant production of H2S in this cell compartment. Detailed characterization of the T-DNA insertion mutants des1-1 and des1-2 has provided insight into the role of sulfide metabolically generated in the cytosol as a signaling molecule. Mutations of L-CYS DESULFHYDRASE 1 (DES1) impede H2S generation in the Arabidopsis cytosol and strongly affect plant metabolism. Senescence-associated vacuoles are detected in mesophyll protoplasts of des1 mutants. Additionally, DES1 deficiency promotes the accumulation and lipidation of the ATG8 protein, which is associated with the process of autophagy. The transcriptional profile of the des1-1 mutant corresponds to its premature senescence and autophagy-induction phenotypes, and restoring H2S generation has been shown to eliminate the phenotypic defects of des1 mutants. Moreover, sulfide is able to reverse ATG8 accumulation and lipidation, even in wild-type plants when autophagy is induced by carbon starvation, suggesting a general effect of sulfide on autophagy regulation that is unrelated to sulfur or nitrogen limitation stress. Our results suggest that cysteine-generated sulfide in the cytosol negatively regulates autophagy and modulates the transcriptional profile of Arabidopsis.

The target of rapamycin (TOR) kinase integrates nutritional and stress signals to coordinately control cell growth in all eukaryotes. TOR associates with highly conserved proteins to constitute two distinct signaling complexes termed TORC1 and TORC2. Inactivation of TORC1 by rapamycin negatively regulates protein synthesis in most eukaryotes. Here, we report that down-regulation of TOR signaling by rapamycin in the model green alga Chlamydomonas reinhardtii resulted in pronounced phosphorylation of the endoplasmic reticulum chaperone BiP. Our results indicated that Chlamydomonas TOR regulates BiP phosphorylation through the control of protein synthesis, since rapamycin and cycloheximide have similar effects on BiP modification and protein synthesis inhibition. Modification of BiP by phosphorylation was suppressed under conditions that require the chaperone activity of BiP, such as heat shock stress or tunicamycin treatment, which inhibits N-linked glycosylation of nascent proteins in the endoplasmic reticulum. A phosphopeptide localized in the substrate-binding domain of BiP was identified in Chlamydomonas cells treated with rapamycin. This peptide contains a highly conserved threonine residue that might regulate BiP function, as demonstrated by yeast functional assays. Thus, our study has revealed a regulatory mechanism of BiP in Chlamydomonas by phosphorylation/dephosphorylation events and assigns a role to the TOR pathway in the control of BiP modification.

Nos propusimos utilizar el alga verde unicelular Chlamydomonas reinhardtii como modelo para el estudio de la cascada de señalización TOR en organismos fotosintéticos. Así, al inicio de esta tesis nos planteamos los siguientes objetivos:1. Análisis de la sensibilidad del organismo fotosintético Chlamydomonas reinhardtii al tratamiento con rapamicina, droga inhibidora de la quinasa TOR.2. Identifica ción, clonación y caracterización de los genes TOR y FKBP12 en Chlamydomonas.3. Identificación y caracterización de proteínas asociadas a TOR en Chlamydomonas.4. Caracterización de los efectos fisiológicos provocados por la inhibición de TOR en Chlamydomonas.