RESUMEN
Abscisic acid (ABA) regulates numerous developmental processes and drought tolerance in plants. Calcium-dependent protein kinases (CPKs) are important Ca2+ sensors playing crucial roles in plant growth and development as well as responses to stresses. However, the molecular mechanisms of many CPKs in ABA signaling and drought tolerance remain largely unknown. Here we combined protein interaction studies, and biochemical and genetic approaches to identify and characterize substrates that were phosphorylated by CPK6 and elucidated the mechanism that underlines the role of CPK6 in ABA signaling and drought tolerance. The expression of CPK6 is induced by ABA and dehydration. Two cpk6 T-DNA insertion mutants are insensitive to ABA during seed germination and root elongation of seedlings; in contrast, overexpression of CPK6 showed the opposite phenotype. Moreover, CPK6-overexpressing lines showed enhanced drought tolerance. CPK6 interacts with and phosphorylates a subset of core ABA signaling-related transcription factors, ABA-responsive element-binding factors (ABFs/AREBs), and enhances their transcriptional activities. The phosphorylation sites in ABF3 and ABI5 were also identified through MS and mutational analyses. Taken together, we present evidence that CPK6 mediates ABA signaling and drought tolerance through phosphorylating ABFs/AREBs. This work thus uncovers a rather conserved mechanism of calcium-dependent Ser/Thr kinases in ABA signaling.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/fisiología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Regulación de la Expresión Génica de las Plantas , Transducción de Señal/genética , Ácido Abscísico/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Sequías , FosforilaciónRESUMEN
Reactive oxygen species (ROS) play important roles in plant growth, development, responses to abiotic and biotic stresses. Hypersensitive response (HR)-like cell death is often associated with excess ROS. However, how a calcium-dependent protein kinase (CPK) modulates this process remains elusive in rapeseed (Brassica napus L.). In the present study, we identified and characterized CPK6L from rapeseed as a novel regulator of ROS and cell death. The subcellular localization of BnaCPK6L was investigated through GFP and was found to be located at the endoplasmic reticulum membrane. Overexpression of the constitutively active BnaCPK6LCA resulted in significant accumulation of ROS and HR-like cell death than the full-length. A quantitative RT-PCR survey identified that the expression levels of a few ROS, cell death and defense-related marker genes were up-regulated upon BnaCPK6LCA expression. Mating-based split ubiquitin system (mbSUS) screening revealed that BnaCPK6L interacted with BnaRBOHD (Respiratory Burst Oxidase Homolog D), which was validated by bimolecular fluorescence complementation (BiFC). An in vitro phosphorylation assay indicated that BnaCPK6L phosphorylated BnaRBOHD. Lastly, we also found that three 2C type protein phosphatases (PP2Cs) interacted with BnaCPK6L. Taken together, this study indicates that BnaCPK6L plays an important role in ROS and HR-like cell death through interacting with and phosphorylating RBOHD.
Asunto(s)
Brassica napus/metabolismo , NADPH Oxidasas/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Brassica napus/genética , Muerte Celular/genética , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica de las Plantas , NADPH Oxidasas/genética , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Unión Proteica , Proteínas Quinasas/genética , Proteína Fosfatasa 2C/genética , Proteína Fosfatasa 2C/metabolismoRESUMEN
Intein is a part of polypeptide in the premature protein with the capability of self-splicing, which is widely applied in protein purification, protein conjuction, cyclopeptide preparation, protein labeling and biosensor. In this review, we summarized the development of intein used in protein purification, discussed intein-mediated chromatographic and non-chromatographic purification systems, and summarized the researches in manipulating intein cleavage reaction. This work is to provide clues for improvement of intein-mediated protein purification.