STIL, a peculiar molecule from styles, specifically dephosphorylates the pollen receptor kinase LePRK2 and stimulates pollen tube growth in vitro

<p>Abstract</p> <p>Background</p> <p>LePRK1 and LePRK2 are two pollen receptor kinases localized to the plasma membrane, where they are present in a high molecular weight complex (LePRK complex). LePRK2 is phosphorylated in mature and germinated pollen, but is dephosphorylated when pollen membranes are incubated with tomato or tobacco style extracts.</p> <p>Results</p> <p>Here we show that LePRK2 dephosphorylation is mediated by a heat-, acid-, base-, DTT- and protease-resistant component from tobacco styles. Using LePRK2 phosphorylation as a tracking assay for purification, style exudates were subjected to chloroform extraction, anionic exchange, and C18 reverse-phase chromatography columns. We finally obtained a single ~3,550 Da compound (as determined by UV-MALDI-TOF MS) that we named STIL (for <b>St</b>yle <b>I</b>nteractor for <b>L</b>ePRKs). STIL increased pollen tube lengths of <it>in vitro </it>germinated pollen in a dose-dependent manner.</p> <p>Conclusion</p> <p>We propose that the LePRK complex perceives STIL, resulting in LePRK2 dephosphorylation and an increase in pollen tube growth.</p

Proteínas G asociadas a sistemas de fototransducción

Fil: Muschietti, Jorge P.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Light signalling transduction by photoreceptors and its implication

El desarrollo de las plantas es un proceso que ocurre bajo fluctuaciones del ambiente lumínico percibidas por fotorreceptores, principalmente los fitocromos A y B (phyA phyB) y los criptocromos 1 y 2 (cry1 y cry2). El estudio de las respuestas en mutantes simples y múltiples de los fotorreceptores es un herramienta muy útil para analizar las vías de señalización. Una parte de nuestro trabajo muestra que el cry1 y la subunidad alpha de la proteína G heterotrimérica (GPA1) de Arabidopsis thaliana interaccionan en la regulación de la apertura del gancho apical en oscuridad y en la acumulación de antocianas en luz azul. cry1 y GPA1 interaccionan genéticamente y plantas mutantes cry1 tienen reducida la unión a GTP γS35. Estudios acerca de los niveles de proteínas sugieren la posible modificación post traduccional de GPA1 mediada por cry1. Por otro lado, estudios del proteoma del cuádruple mutante phyA phyB cry1 cry2 muestran que se encuentran sobre expresadas proteínas involucradas a la respuesta de estrés por temperatura. Con el objetivo de comprender el comportamiento de los mutantes deficientes en fotorreceptores a un shock a altas o bajas temperaturas analizamos además el contenido de ácidos grasos insaturados en sus membranas. Plantas que carecen al menos de phyB muestran una reducción significativa en la proporción de ácidos grasos insaturados de las membranas y esto fue consistente con una reducción en la expresión de los genes que codifican para diferentes desaturasas (FAD2, FAD5, FAD6, FAD7 y FAD 8). Las plantas mutantes para phyB son significativamente más tolerantes a un shock a altas temperaturas, con menor daño en el aparato fotosintético. En condiciones deficientes de luz, la menor instauración de los ácidos grasos de las membranas tilacoidales podría proteger a la planta de un daño mayor en la maquinaria fotosintética frente a un aumento importante de la temperatura ambiental. Estudios comparativos con plantas previamente aclimatadas a altas o bajas temperaturas ayudarán a entender dicha regulación

Protein profiles construction and differential expressed proteins of the Arabidopsis thaliana quadruple mutant phyA phyB cry1 cry2.

En Arabidopsis phyA phyB cry1 y cry2, son los cuatro fotorreceptores más importantes que controlan el crecimiento y desarrollo por la luz. La técnica de geles bidimensionales provee información sobre la abundancia de una proteína y sus modificaciones postraduccionales. Con el objetivo de identificar nuevos componentes en la fototrasnducción de señales estudiamos el perfil proteómico del cuádruple mutante phyA phyB cry1 cry2 (tet). Para esto obtuvimos y comparamos los proteomas específicos del genotipo salvaje (WT) y cuádruple mutante tet de plántulas de Arabidopsis crecidas durante diez días a la luz y en respuesta a la adición o no de sacarosa. En un ensayo paralelo estudiamos únicamente las diferencias en el fosfoproteoma de ambos genotipos con el objetivo de estudiar modificaciones postraduccionales como fosforilación

Adaptative Mechanisms of Striatal Dl andD2 Dopamine Receptors in Response to a Prolonged Reserpine Treatment in Mice

ABSTRACT Mice receiving reserpine (1 mg/kg/day) during 5 days develop behavioral supersensitivity. To study the possible molecular cor relatesofthese adaptative changes we compared binding param eters of Dl and 02 receptors and adenylate cyclase activity in stnata from normal and reserpinized mice. Saturation curves using [3H]SCH 23390 showed no changes in maximum binding capacity (B@,) Behavioral, electrophysiological and biochemical studies in dicate that long-term administration of neuroleptics (Rupnia

cry1 and GPA1 signaling genetically interact in hook opening and anthocyanin synthesis in Arabidopsis.

While studying blue light-independent effects of cryptochrome 1 (cry1) photoreceptor, we observed premature opening of the hook in cry1 mutants grown in complete darkness, a phenotype that resembles the one described for the heterotrimeric G-protein α subunit (GPA1) null mutant gpa1. Both cry1 and gpa1 also showed reduced accumulation of anthocyanin under blue light. These convergent gpa1 and cry1 phenotypes required the presence of sucrose in the growth media and were not additive in the cry1 gpa1 double mutant, suggesting context-dependent signaling convergence between cry1 and GPA1 signaling pathways. Both, gpa1 and cry1 mutants showed reduced GTP-binding activity. The cry1 mutant showed wild-type levels of GPA1 mRNA or GPA1 protein. However, an anti-transducin antibody (AS/7) typically used for plant Gα proteins, recognized a 54 kDa band in the wild type but not in gpa1 and cry1 mutants. We propose a model where cry1-mediated post-translational modification of GPA1 alters its GTP-binding activity

STIL, a peculiar molecule from styles, specifically dephosphorylates the pollen receptor kinase LePRK2 and stimulates pollen tube growth in vitro.

BackgroundLePRK1 and LePRK2 are two pollen receptor kinases localized to the plasma membrane, where they are present in a high molecular weight complex (LePRK complex). LePRK2 is phosphorylated in mature and germinated pollen, but is dephosphorylated when pollen membranes are incubated with tomato or tobacco style extracts.ResultsHere we show that LePRK2 dephosphorylation is mediated by a heat-, acid-, base-, DTT- and protease-resistant component from tobacco styles. Using LePRK2 phosphorylation as a tracking assay for purification, style exudates were subjected to chloroform extraction, anionic exchange, and C18 reverse-phase chromatography columns. We finally obtained a single ~3,550 Da compound (as determined by UV-MALDI-TOF MS) that we named STIL (for Style Interactor for LePRKs). STIL increased pollen tube lengths of in vitro germinated pollen in a dose-dependent manner.ConclusionWe propose that the LePRK complex perceives STIL, resulting in LePRK2 dephosphorylation and an increase in pollen tube growth

Proteome analysis of differentially expressed nuclear proteins in the phyA phyB cry1 cry2 quadruple mutant of Arabidopsis thaliana.

Phytochromes phyA and phyB, and cryptochromes cry1 and cry2 are the fourth main photoreceptors that control growth and development in plants by light. These photoreceptors are present in the nucleus and affect the expression levels of transcription factors and nuclear regulatory proteins that interact with light regulated gene promoters. DIGE techniques (Differential In Gel Electrophoresis) provides information not only of the abundance of proteins but also possible postranslational modifications when stained with Pro-Q Diamond. The main purpose of the work is to identify new nuclear proteins regulated by these photoreceptors. We constructed a proteome profile from nuclear enrich fractions of phyA phyB cry1 cry2 quadruple mutant and WT light grown Arabidopsis seedlings. Nuclear protein enrich fractions were obtained by a new modified protocol (based on Folta and Kaufman, 2006 and Calikowski and Meier, 2006)that enriched four times in nuclear proteins. Statistical analysis showed 39 differentially expressed spots where 15 spots were over expressed and 24 spots underexpressed in the quadruple mutant (p< 0.05, FDR 0.05). To study whether any of these 39 proteins were phosphorylated, we analyzed when the intensity of the Cy3+ ProQ Diamond fluorescence signal compared to the Cy3 signal. We found 2 potentially phosphorylated spots. The identity of all these nuclear proteins will provide relevant information toward elucidation on photoreceptor signaling pathways and their regulatory mechanisms

Molecular, biochemical and physiological study of the factors that regulate heat stress tolerance on Arabidopsis thaliana photoreceptors mutant plants

Las plantas son organismos sésiles en ambientes fluctuantes cuya plasticidad fenotípica y capacidad de integrar señales ambientales les permiten ajustar su crecimiento y desarrollo. La luz y la temperatura conforman uno de los factores ambientales más importantes. Varias familias de fotorreceptores perciben la luz. Los fitocromos (phyA-phyE) perciben la luz roja y rojo lejana, y los criptocromos (cry1-cry3) la luz azul y UV-A. En estudios de perfiles proteicos encontramos que un grupo de proteínas involucradas en la termotolerancia estaban sobreexpresadas en plántulas desetioladas del cuádruple mutante phyAphyBcry1cry2 respecto al genotipo salvaje (WT). Un 80% de plántulas WT y doble mutante cry1cry2 resultaron dañadas luego de un shock por calor, mientras que sólo el 15 % de los mutantes phyB y phyAphyBcrycry2 resultaron dañados. La expresión de las desaturasas de ácidos grasos FAD2, FAD5, FAD6, FAD7 y FAD8 fue menor en los genotipos que llevan la mutación phyB. Esto fue consistente con una menor instauración de los de ácidos grasos de sus membranas. Estos resultados sugieren que el phyB controlaría la expresión de las desaturasas a nivel transcripcional y la composición de ácidos grasos insaturados en Arabidopsis. Estamos evaluando la magnitud del efecto del estrés oxidativo en los tratamientos de calor

Cryptochrome 1 and Heterotrimeric G protein regulate common developmental responses in Arabidopsis

Plants sense and interpret the light environment and adjust their growth and development accordingly. In Arabidopsis thaliana blue light is perceived by two cryptochromes (cry1 and cry2). Arabidopsis has a single gene encoding for the alpha subunit of the heterotrimeric G protein (GPA1). Heterotrimeric G proteins have been largely implicated in blue signaling during Arabidopsis seedling development. Here we combine a biochemical, molecular and genetic approach to study signaling pathway connections between GPA1 and cry1. GTP binding activity in the cry1 mutant was significantly reduced to gpa1 mutant levels. GPA transcript or protein levels were normal in the cry1 mutant and in our experimental conditions we did not find protein–protein interaction. cry1 mutants failed in some typical gpa1 mediated responses in darkness and cry1 gpa1 double mutants studies showed genetic epistasis. Microarray experiments showed cry1 and gpa1 coregulate the expression of several genes. Finally, adult cry1 gpa1 double mutants showed altered leaf shape, trichome formation and rosette size. All these results suggest cry1 and gpa1 regulate common processes during Arabidopsis seedling development