RESUMEN
Chromatin is a major determinant in the regulation of virtually all DNA-dependent processes. Chromatin architectural proteins interact with nucleosomes to modulate chromatin accessibility and higher-order chromatin structure. The evolutionarily conserved DEK domain-containing protein is implicated in important chromatin-related processes in animals, but little is known about its DNA targets and protein interaction partners. In plants, the role of DEK has remained elusive. In this work, we identified DEK3 as a chromatin-associated protein in Arabidopsis thaliana. DEK3 specifically binds histones H3 and H4. Purification of other proteins associated with nuclear DEK3 also established DNA topoisomerase 1α and proteins of the cohesion complex as in vivo interaction partners. Genome-wide mapping of DEK3 binding sites by chromatin immunoprecipitation followed by deep sequencing revealed enrichment of DEK3 at protein-coding genes throughout the genome. Using DEK3 knockout and overexpressor lines, we show that DEK3 affects nucleosome occupancy and chromatin accessibility and modulates the expression of DEK3 target genes. Furthermore, functional levels of DEK3 are crucial for stress tolerance. Overall, data indicate that DEK3 contributes to modulation of Arabidopsis chromatin structure and function.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Cromatina/genética , Regulación de la Expresión Génica de las Plantas , Nucleosomas/metabolismo , Secuencia de Aminoácidos , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Calpaína/genética , Calpaína/metabolismo , Cromatina/fisiología , Cromatina/ultraestructura , Histonas/metabolismo , Datos de Secuencia Molecular , Mutación , Estructura Terciaria de Proteína , Proteínas Recombinantes , Alineación de Secuencia , Estrés FisiológicoRESUMEN
BACKGROUND: Plant GSK-3/Shaggy-like kinases are key players in brassinosteroid (BR) signalling which impact on plant development and participate in response to wounding, pathogens and salt stress. Bikinin was previously identified in a chemical genetics screen as an inhibitor targeting these kinases. To dissect the structural elements crucial for inhibition of GSK-3/Shaggy-like kinases by bikinin and to isolate more potent compounds we synthesised a number of related substances and tested their inhibitory activity in vitro and in vivo using Arabidopsis thaliana. RESULTS: A pyridine ring with an amido succinic acid residue in position 2 and a halogen in position 5 were crucial for inhibitory activity. The compound with an iodine substituent in position 5, denoted iodobikinin, was most active in inhibiting BIN2 activity in vitro and efficiently induced brassinosteroid-like responses in vivo. Its methyl ester, methyliodobikinin, showed improved cell permeability, making it highly potent in vivo although it had lower activity in vitro. HPLC analysis revealed that the methyl residue was rapidly cleaved off in planta liberating active iodobikinin. In addition, we provide evidence that iodobikinin and bikinin are inactivated in planta by conjugation with glutamic acid or malic acid and that the latter process is catalysed by the malate transferase SNG1. CONCLUSION: Brassinosteroids participate in regulation of many aspects of plant development and in responses to environmental cues. Thus compounds modulating their action are valuable tools to study such processes and may be an interesting opportunity to modify plant growth and performance in horticulture and agronomy. Here we report the development of bikinin derivatives with increased potency that can activate BR signalling and mimic BR action. Methyliodobikinin was 3.4 times more active in vivo than bikinin. The main reason for the superior activity of methyliodobikinin, the most potent compound, is its enhanced plant tissue permeability. Inactivation of bikinin and its derivatives in planta involves SNG1, which constitutes a novel pathway for modification of xenobiotic compounds.
Asunto(s)
Aminopiridinas/metabolismo , Aminopiridinas/farmacología , Arabidopsis/enzimología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Succinatos/metabolismo , Succinatos/farmacología , Aminopiridinas/química , Arabidopsis/efectos de los fármacos , Proteínas de Arabidopsis/metabolismo , Biocatálisis/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/metabolismo , Hidrólisis/efectos de los fármacos , Hipocótilo/efectos de los fármacos , Hipocótilo/crecimiento & desarrollo , Malatos/metabolismo , Espectrometría de Masas , Metilación/efectos de los fármacos , Permeabilidad/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , Protoplastos/efectos de los fármacos , Protoplastos/metabolismo , Estándares de Referencia , Transducción de Señal/efectos de los fármacos , Espectrofotometría Ultravioleta , Succinatos/químicaRESUMEN
Stomatal formation is regulated by multiple developmental and environmental signals, but how these signals are integrated to control this process is not fully understood. In Arabidopsis thaliana, the basic helix-loop-helix transcription factor SPEECHLESS (SPCH) regulates the entry, amplifying and spacing divisions that occur during stomatal lineage development. SPCH activity is negatively regulated by mitogen-activated protein kinase (MAPK)-mediated phosphorylation. Here, we show that in addition to MAPKs, SPCH activity is also modulated by brassinosteroid (BR) signalling. The GSK3/SHAGGY-like kinase BIN2 (BR INSENSITIVE2) phosphorylates residues overlapping those targeted by the MAPKs, as well as four residues in the amino-terminal region of the protein outside the MAPK target domain. These phosphorylation events antagonize SPCH activity and limit epidermal cell proliferation. Conversely, inhibition of BIN2 activity in vivo stabilizes SPCH and triggers excessive stomatal and non-stomatal cell formation. We demonstrate that through phosphorylation inputs from both MAPKs and BIN2, SPCH serves as an integration node for stomata and BR signalling pathways to control stomatal development in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Brasinoesteroides/metabolismo , Estomas de Plantas/metabolismo , Transducción de Señal , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FosforilaciónRESUMEN
Brassinosteroids (BRs) are plant hormones that regulate many processes including cell elongation, leaf development, pollen tube growth and xylem differentiation. GSK3/shaggy-like kinases (GSK) are critical regulators of intracellular signalling initiated by the binding of BR to the BRI1 receptor complex. Three GSKs have already been shown to relay BR responses, including phosphorylation of the transcriptional regulator BES1. However, recent studies indicate that one or more yet unidentified protein kinases are involved in BR signalling. Here, we show that the in vivo protein kinase activity of the group-III GSK, ASKtheta, was negatively regulated by BRI1. Arabidopsis thaliana plants with enhanced ASKtheta activity displayed a bri1-like phenotype. ASKtheta overexpressors accumulated high levels of brassinolide, castasterone and typhasterol, and were insensitive to BR. ASKtheta localized to the nucleus and directly phosphorylated BES1 and BZR1. Moreover, the BES1/BZR1-like transcription factor BEH2 was isolated as an ASKtheta interaction partner in a yeast two-hybrid screen. ASKtheta phosphorylated BEH2 both in vitro and in vivo. Overall, these data provide strong evidence that ASKtheta is a novel component of the BR signalling cascade, targeting the transcription factors BES1, BZR1 and BEH2.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas Quinasas/metabolismo , Transducción de Señal , Arabidopsis/enzimología , Proteínas de Arabidopsis/genética , Brasinoesteroides , Colestanoles/metabolismo , Proteínas de Unión al ADN , Regulación de la Expresión Génica de las Plantas , Proteínas Nucleares/metabolismo , Fosforilación , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Proteínas Quinasas/genética , ARN de Planta/genética , Esteroides Heterocíclicos/metabolismoRESUMEN
Glycogen synthase kinase 3 (GSK3) is a key regulator in signaling pathways in both animals and plants. Three Arabidopsis thaliana GSK3s are shown to be related to brassinosteroid (BR) signaling. In a phenotype-based compound screen we identified bikinin, a small molecule that activates BR signaling downstream of the BR receptor. Bikinin directly binds the GSK3 BIN2 and acts as an ATP competitor. Furthermore, bikinin inhibits the activity of six other Arabidopsis GSK3s. Genome-wide transcript analyses demonstrate that simultaneous inhibition of seven GSK3s is sufficient to activate BR responses. Our data suggest that GSK3 inhibition is the sole activation mode of BR signaling and argues against GSK3-independent BR responses in Arabidopsis. The opportunity to generate multiple and conditional knockouts in key regulators in the BR signaling pathway by bikinin represents a useful tool to further unravel regulatory mechanisms.
Asunto(s)
Aminopiridinas/farmacología , Arabidopsis/enzimología , Colestanoles/farmacología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Esteroides Heterocíclicos/farmacología , Succinatos/farmacología , Secuencia de Aminoácidos , Aminopiridinas/química , Proteínas de Arabidopsis/antagonistas & inhibidores , Proteínas de Arabidopsis/metabolismo , Brasinoesteroides , Colestanoles/química , Proteínas de Unión al ADN , Glucógeno Sintasa Quinasa 3/metabolismo , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Fosforilación , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Proteínas Quinasas/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Esteroides Heterocíclicos/química , Succinatos/químicaRESUMEN
The DNA of many eukaryotes is methylated at specific cytosine residues in connection with gene regulation. Here we report a method for the quantification of global cytosine methylation based on enzymatic hydrolysis of DNA, dephosphorylation, and subsequent high-performance cation exchange chromatography. Nucleosides are separated in less than 3 min under isocratic conditions on a benzenesulfonic acid-modified silica phase and detected by UV absorption. As little as 1 microg of DNA is sufficient to measure 5-methyldeoxycytosine levels with a typical relative standard deviation of less than 3%. As a proof of concept, the method was applied for analysis of DNA from several Arabidopsis thaliana mutants affected in DNA methylation and from Medicago sativa seedlings treated with the environmental pollutant chromium(VI).