Participación del gen AtCPK1 en la defensa de Arabidopsis thaliana frente a patógenos

[spa] Las plantas han desarrollado mecanismos sofisticados de defensa para protegerse de los organismos perjudiciales de su entorno. La resistencia depende de múltiples mecanismos de protección que comprenden, tanto barreras físicas y químicas constitutivas, como respuestas inducibles. Estas son activadas ante el reconocimiento de la presencia del patógeno que desencadena en la planta una serie de reacciones defensivas que incluyen una reprogramación transcripcional de los genes de defensa, la producción de metabolitos secundarios (fitoalexinas), el reforzamiento de la pared celular y en algunos casos la muerte celular programada, con el fin de evitar la propagación del patógeno. Uno de los tópicos más importante de investigación en plantas y al cual se están dedicando grandes esfuerzos es entender los procesos de señalización que llevan a la activación de las respuestas de defensa en las plantas, por su particular relevancia en la protección de cultivos de todo del mundo. Las reacciones de defensa tempranas desencadenadas por la percepción del patógeno en las células vegetales comprenden la despolarización de la membrana plasmática, cambios en los niveles de calcio intracelular, la producción de especies reactivas de oxígeno (ROS), seguidos por la activación de proteínas quinasas y los consiguientes procesos de fosforilación reversible de proteínas reguladas por calcio. Las plantas poseen diversas clases de proteínas sensoras de calcio, entre ellas las proteínas quinasas dependientes de calcio (CPKs o CDPKs). La peculiaridad de estas proteínas reside en que en la misma cadena polipeptídica encontramos el dominio quinasa, un dominio autorregulador y el dominio calmodulina de unión a Ca2+, pudiendo ser activadas directamente por la unión del Ca2+. Esta estructura tan particular de las CPKs las convierte en sensores de cambios en los niveles de calcio y transductores en actividad quinasa de proteínas que median los procesos de señalización posteriores. El trabajo de este proyecto de tesis doctoral ha estado dirigido al estudio y la caracterización de la participación de una proteína CPK de la planta modelo Arabidopsis thaliana, la proteína AtCPK1, en la defensa frente a patógenos. Al inicio del trabajo, se disponía de evidencias preliminares obtenidas en el grupo de investigación que demostraban la implicación funcional de esta proteína AtCPK1 en la resistencia de la planta a infección por patógenos, lo que justificaba el interés de este estudio. Los estudios desarrollados en este trabajo demuestran que la expresión del gen AtCPK1 se induce rápidamente en respuesta a la infección por el hongo patógeno Fusarium oxysporum y al tratamiento con elicitores derivados del mismo hongo en plantas de Arabidopsis. Además de la regulación transcripcional, se ha mostrado que la correspondiente proteína está regulada postraduccionalmente. Así, los niveles de acumulación de la proteína AtCPK1 están controlados por la vía de degradación de proteínas dependiente del proteasoma. También se ha observado que la proteína AtCPK1 presenta distintos estados de fosforilación, y en respuesta a la infección fúngica la proteína se fosforila bien por autofosforilación, bien por otras quinasas pendientes de indentificación. Por otra parte, la presencia de iones calcio determina de manera importante la capacidad de interacción de AtCPK1 con otras proteínas, mimetizando el efecto de la infección en relación a las proteínas que interaccionan con AtCPK1. Con el objetivo de entender mejor la participación de AtCPK1 en la respuesta de defensa de la planta y de identificar otros componentes implicados en las rutas de transducción de señales activadas por infección, en este trabajo se ha caracterizado el interactoma de AtCPK1 mediante diferentes estrategias complementarias. Entre las proteínas identificadas que interaccionan con AtCPK1 o asociadas a los complejos multiproteicos en los que participa AtCPK1 se encuentran proteínas de la familia de las 14-3-3, enzimas implicadas en la protección frente a estrés oxidativo (ascorbato peroxidasa y catalasas) y en la detoxificación celular (nitrilasas), así como también proteínas cloroplásticas que participan en el ciclo oxidativo del cloroplasto (la ATP sintasa y proteínas de unión a las clorofilas). Estos estudios además han mostrado una localización cloroplástica de la proteína AtCPK1, añadiendo un nivel de complejidad más en cuanto al compartimento subcelular en el cual AtCPK1 desempeña su función y se añade a las localizaciones anteriormente descritas para esta proteína en peroxisomas y cuerpos lipídicos de las células de Arabidopsis

Proteasome-associated ubiquitin ligase relays target plant hormone-specific transcriptional activators

The ubiquitin-proteasome system is vital to hormone-mediated developmental and stress responses in plants. Ubiquitin ligases target hormone-specific transcriptional activators (TAs) for degradation, but how TAs are processed by proteasomes remains unknown. We report that in Arabidopsis, the salicylic acid– and ethylene-responsive TAs, NPR1 and EIN3, are relayed from pathway-specific ubiquitin ligases to proteasome-associated HECT-type UPL3/4 ligases. Activity and stability of NPR1 were regulated by sequential action of three ubiquitin ligases, including UPL3/4, while proteasome processing of EIN3 required physical handover between ethylene-responsive SCF(EBF2) and UPL3/4 ligases. Consequently, UPL3/4 controlled extensive hormone-induced developmental and stress-responsive transcriptional programs. Thus, our findings identify unknown ubiquitin ligase relays that terminate with proteasome-associated HECT-type ligases, which may be a universal mechanism for processive degradation of proteasome-targeted TAs and other substrates

Harnessing the ubiquitin code to respond to environmental cues

Ubiquitination is an essential post-translational signal that allows cells to adapt and respond to environmental stimuli. Substrate modifications range from a single ubiquitin molecule to complex polyubiquitin chains, where diverse chain topologies constitute a code that is utilized to modify the functions of proteins in numerous cellular signalling pathways. Diverse ubiquitin chain topologies are generated by linking the C-terminus of ubiquitin to one of seven lysine residues or the N-terminal methionine 1 residue of the preceding ubiquitin. Cooperative action between a large array of E2 conjugating and E3 ligase enzymes supports the formation of not only homotypic ubiquitin chains but also heterotypic mixed or branched chains. This complex array of chain topologies is recognized by proteins containing linkage-specific ubiquitin-binding domains and regulates numerous cellular pathways. Although many functions of the ubiquitin code in plants remain unknown, recent work suggests that specific chain topologies are associated with particular molecular processes. Deciphering the ubiquitin code and how plants utilize it to cope with the changing environment is essential to understand the regulatory mechanisms that underpin myriad stress responses and establishment of environmental tolerance

The autotaxin-lysophosphatidic acid pathway in pathogenesis of rheumatoid arthritis

[EN] Lysophosphatidicacid (LPA )is a phospholipid that i smainly produced by the hydrolysis of lysopho- sphatidylcholine (LPC) by lysophospholipase D, which is also called autotaxin (ATX).LPA interacts with specific G-protein coupled receptors and is involved in the regulation of cellular survival, proliferation,differentiation and motility . LPA also has roles in several pathological disorders, such as cancer and pulmonary, dermal and renal fibrosis. The involvement of the ATX?LPA pathway has recently been demonstrated in inflammatory responses and apoptosis of fibroblast like synoviocytes (FLS) from patients with rheumatoid arthritis and during the development of experimental arthritis.This review summarises the current literature of the ATX?LPA pathway in rheumatoid arthritis.Instituto de Salud Carlos III (ISCIII)European Regional Development Fund of the European Unio

SUMO Conjugation to BZR1 Enables Brassinosteroid Signaling to Integrate Environmental Cues to Shape Plant Growth

Brassinosteroids (BRs) play crucial roles in plant development, but little is known of mechanisms that integrate environmental cues into BR signaling. Conjugation to the small ubiquitin-like modifier (SUMO) is emerging as an important mechanism to transduce environmental cues into cellular signaling. In this study, we show that SUMOylation of BZR1, a key transcription factor of BR signaling, provides a conduit for environmental influence to modulate growth during stress. SUMOylation stabilizes BZR1 in the nucleus by inhibiting its interaction with BIN2 kinase. During salt stress, Arabidopsis plants arrest growth through deSUMOylation of BZR1 in the cytoplasm by promoting the accumulation of the BZR1 targeting SUMO protease, ULP1a. ULP1a mutants are salt tolerant and insensitive to the BR inhibitor, brassinazole. BR treatment stimulates ULP1a degradation, allowing SUMOylated BZR1 to accumulate and promote growth. This study uncovers a mechanism for integrating environmental cues into BR signaling to shape growth

Effect of lysophosphatidic acid receptor inhibition on bone changes in ovariectomized mice

[EN] Pharmacological inhibition of signaling through lysophosphatidic acid (LPA) receptors reduces bone erosions in an experimental model of arthritis by mechanisms involving reduced osteoclast differentiation and bone resorption and increased differentiation of osteoblasts and bone mineralization. These results led us to hypothesize that LPA receptor inhibition would be beneficial in osteoporosis. Our aim was to test this hypothesis with the LPA receptor antagonist, Ki16425, in ovariectomized mice, a model of postmenopausal osteoporosis. Ovariectomized mice treated with Ki16425 showed bone loss similar to that observed in the controls. Osteoblast markers, Alpl, Bglap and Col1a1, were increased at the mRNA level but no changes were detected in serum. No additional difference was observed in the Ki16425-treated mice relative to the ovariectomized controls with regard to osteoclast function markers or assays of matrix mineralization or osteoclast differentiation. Thus, pharmacological inhibition of LPA receptor was not beneficial for preventing bone loss in ovariectomized mice, indicating that its favorable effect on bone remodeling is less general than hypothesized

Phosphoproteome analyses pinpoint the F‐box protein SLOW MOTION as a regulator of warm temperature‐mediated hypocotyl growth in Arabidopsis

Hypocotyl elongation is controlled by several signals and is a major characteristic of plantsgrowing in darkness or under warm temperature. While already several molecular mechan-isms associated with this process are known, protein degradation and associated E3 ligaseshave hardly been studied in the context of warm temperature.In a time-course phosphoproteome analysis onArabidopsisseedlings exposed to control orwarm ambient temperature, we observed reduced levels of diverse proteins over time, whichcould be due to transcription, translation, and/or degradation. In addition, we observed dif-ferential phosphorylation of the LRR F-box protein SLOMO MOTION (SLOMO) at two ser-ine residues.We demonstrate that SLOMO is a negative regulator of hypocotyl growth, also underwarm temperature conditions, and protein–protein interaction studies revealed possible inter-actors of SLOMO, such as MKK5, DWF1, and NCED4. We identified DWF1 as a likelySLOMO substrate and a regulator of warm temperature-mediated hypocotyl growth.We propose that warm temperature-mediated regulation of SLOMO activity controls theabundance of hypocotyl growth regulators, such as DWF1, through ubiquitin-mediateddegradatio

Root branching toward water involves posttranslational modification of transcription factor ARF7

Plants adapt to heterogeneous soil conditions by altering their root architecture. For example, roots branch when in contact with water using the hydropatterning response. We report that hydropatterning is dependent on auxin response factor ARF7. This transcription 5 factor induces asymmetric expression of its target gene LBD16 in lateral root founder cells on the side of the root in contact with water. This differential expression pattern is regulated by post-translational modification of ARF7 with the SUMO protein. SUMOylation negatively regulates ARF7 DNA binding activity. ARF7 SUMOylation is required to recruit the Aux/IAA repressor protein IAA3. Blocking ARF7 SUMOylation disrupts IAA3 10 recruitment and hydropatterning. We conclude that SUMO-dependent regulation of auxin response controls root branching pattern in response to water availability