RESUMEN
BACKGROUND: Maize is a major cereal crop world widely, however, the yield of maize is frequently limited by dehydration and even death of plants, which resulted from osmotic stress such as drought and salinity. Dissection of molecular mechanisms controlling stress tolerance will enable plant scientists and breeders to increase crops yield by manipulating key regulatory components. METHODS: The candidate OSR1 gene was identified by map-based cloning. The expression level of OSR1 was verified by qRT-PCR and digital PCR in WT and osr1 mutant. Electrophoretic mobility shift assay, transactivation activity assay, subcellular localization, transcriptome analysis and physiological characters measurements were conducted to analyze the function of OSR1 in osmotic stress resistance in maize. RESULTS: The osr1 mutant was significantly less sensitive to osmotic stress than the WT plants and displayed stronger water-holding capacity, and the OSR1 homologous mutant in Arabidopsis showed a phenotype similar with maize osr1 mutant. Differentially expressed genes (DEGs) were identified between WT and osr1 under osmotic stress by transcriptome analysis, the expression levels of many genes, such as LEA, auxin-related factors, PPR family members, and TPR family members, changed notably, which may primarily involve in osmotic stress or promote root development. CONCLUSIONS: OSR1 may serve as a negative regulatory factor in response to osmotic stress in maize. The present study sheds new light on the molecular mechanisms of osmotic stress in maize.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Presión Osmótica , Proteínas de Plantas , Factores de Transcripción , Zea mays , Zea mays/genética , Zea mays/metabolismo , Zea mays/fisiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Mutación , Estrés Fisiológico/genética , Perfilación de la Expresión GénicaRESUMEN
Plasma membrane (PM)-associated abscisic acid (ABA) signal transduction is an important component of ABA signaling. The C2-domain ABA-related (CAR) proteins have been reported to play a crucial role in recruiting ABA receptor PYR1/PYL/RCAR (PYLs) to the PM. However, the molecular details of the involvement of CAR proteins in membrane-delimited ABA signal transduction remain unclear. For instance, where this response process takes place and whether any additional members besides PYL are taking part in this signaling process. Here, the GUS-tagged materials for all Arabidopsis CAR members were used to comprehensively visualize the extensive expression patterns of the CAR family genes. Based on the representativeness of CAR1 in response to ABA, we determined to use it as a target to study the function of CAR proteins in PM-associated ABA signaling. Single-particle tracking showed that ABA affected the spatiotemporal dynamics of CAR1. The presence of ABA prolonged the dwell time of CAR1 on the membrane and showed faster lateral mobility. Surprisingly, we verified that CAR1 could directly recruit hypersensitive to ABA1 (HAB1) and SNF1-related protein kinase 2.2 (SnRK2.2) to the PM at both the bulk and single-molecule levels. Furthermore, PM localization of CAR1 was demonstrated to be related to membrane microdomains. Collectively, our study revealed that CARs recruited the three main components of ABA signaling to the PM to respond positively to ABA. This study deepens our understanding of ABA signal transduction.
Asunto(s)
Ácido Abscísico , Proteínas de Arabidopsis , Arabidopsis , Membrana Celular , Proteínas Serina-Treonina Quinasas , Transducción de Señal , Ácido Abscísico/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Regulación de la Expresión Génica de las Plantas , Reguladores del Crecimiento de las Plantas/metabolismo , Plantas Modificadas Genéticamente , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Single-molecule imaging is emerging as a revolutionary approach to studying fundamental questions in plants. However, compared with its use in animals, the application of single-molecule imaging in plants is still underexplored. Here, we review the applications, advantages, and challenges of single-molecule fluorescence imaging in plant systems from the perspective of methodology. Firstly, we provide a general overview of single-molecule imaging methods and their principles. Next, we summarize the unprecedented quantitative details that can be obtained using single-molecule techniques compared to bulk assays. Finally, we discuss the main problems encountered at this stage and provide possible solutions.
Asunto(s)
Fenómenos Fisiológicos de las Plantas , Plantas/metabolismo , Imagen Individual de Molécula/métodosRESUMEN
Glutathione peroxidases (GPXs) regulate the levels of reactive oxygen species in cells and tissues. During the redox cycling, the plant GPX is regenerated by thioredoxins (TRXs) as reductant rather than glutathione as the electron donor. However, the direct experimental observation on the interaction dynamics between GPXs and TRXs has not been reported, and the redox mechanism is unclear. In this work, the protein interactions between oxidized AtGPX3 and reduced AtTRXh9 have been studied using single-molecule fluorescence resonance energy transfer (smFRET). The obtained results indicate there are four processes in these two protein interaction, including biological recognition, binding, intermediate and unbinding state. Two enzymatic reaction intermediate states have been identified in the dissociation of AtGPX3-AtTRXh9 complex from binding to unbinding state, suggesting two types of interaction pathways and intermediate complexes. In particular, the dynamical study reveals that the redox reaction between oxidized AtGPX3 and reduced AtTRXh9 is realized through the forming and breaking of disulfide bonds via the active sites of Cys4 and Cys57 in AtTRXh9. These findings are of significant for deep understanding the redox reaction and mechanism between GPXs and TRXs enzymes, and studying other protein dynamics at single-molecule level.
Asunto(s)
Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis , Glutatión/metabolismo , Glutatión Peroxidasa/metabolismo , Oxidación-Reducción , Tiorredoxinas/metabolismoRESUMEN
The cellular redox states directly affect cell proliferation, differentiation and apoptosis, and the redox states changes is particularly important to the regulation of cell survival or death. Thioredoxin is a kind of oxidation regulatory protein which is widely exists in organisms, and the change of redox states is also an important process in redox regulation. In this work, we have used the site-directed mutagenesis of protein, SDS-polyacrylamide gel electrophoresis fluorescence spectroscopy and circular dichroism etc., to investigate redox states changes between TRX (E. coli) and glutathione peroxidase(GPX3) during their interaction. By observing the fluorescence spectra of TRX and its mutants, we have studied the protein interactions as well as the redox states switching between oxidation state TRX and the reduced state GPX3. The results demonstrate the presence of interactions and electron exchanges occurring between reduced GPX3 and oxidized TRX, which is of significance for revealing the physical and chemical mechanism of TRX in intracellular signal transduction.