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Cryptochromes (CRYs) belong to an ancient and conserved class of blue-light receptor regulating circadian clock and development in animals and plants. Arabidopsis CRY2 form physiologically active homodimers in response to blue light treatment and further oligomerize into photobodies, which are expected to be the foci harboring protein interaction, phosphorylation, and ubiquitination. Here we describe two efficient methods developed to test the formation of blue-light-dependent photobodies of CRY-GFP fusing proteins using the mesophyll protoplasts of Arabidopsis or soybean, respectively.Seedling deetiolation is a hallmark of the photomorphogenic response, and successful conversion of protochlorophyllide (Pchlide) into chlorophyllide during initial light exposure is critical for plant survival and growth. Here we describe the seedling deetiolation process of two typical mutants pif3 and flu by analysis of the cotyledons greening, Pchlide content, and reactive oxygen species (ROS) production and summarize a set of general methods for the research of seedling greening.The UV RESISTANCE LOCUS 8 (UVR8) is a photoreceptor mediating photomorphogenic responses to UV-B. UVR8 exists as homodimer in plants and UV-B induces dissociation of dimeric UVR8 into monomers to initiate responses. The monomer/dimer status of UVR8 is reversible and a dynamic photo-equilibrium is established in plants according to the ambient light conditions. Here we describe a method to detect UVR8 homodimer and monomer by immunoblotting method from tomato (Solanum lycopersicum) plants. The feature of this method is that protein samples are not boiled prior to loading on an SDS-PAGE gel, which allows the detection of UVR8 homodimer and monomers simultaneously with a single antibody.The red (R)/far-red (FR) light absorbing phytochromes are one of the major photoreceptor classes in plants. Phytochromes exist in two distinct but interconvertible forms the R light-absorbing Pr form and the FR light-absorbing Pfr form. Upon photoactivation by light, phytochromes physically interact with their partners to transduce the light signal. Co-immunoprecipitation (Co-IP) is one of the most efficient techniques to study these protein-protein interactions in vivo. However, the co-IP procedure for phytochromes needs to be modified to allow their formation of Pr or Pfr form. Here, we describe a detailed co-IP procedure to examine which form of phytochrome (Pr or Pfr) is preferentially associated with their interacting partners in vivo.The circadian clock is a self-sustaining 24 h timekeeper which enables plants to anticipate periodic environmental changes and optimize the biological activities for most beneficial time during day/night cycles. EGCG in vitro As in many organisms, the sustained circadian rhythmicity in plant relies on network of transcriptional/translational feedback loops (TTFLs) of transcription factors at the core of the oscillator. Over the past years, ChIP-seq has become an indispensable method to uncover the clock network through identifications of circadian transcription factors binding sites on a genome-wide scale. Here, we show how to use ChIP-seq to analyze the occupancy of circadian transcription factor in Arabidopsis. In addition, we briefly describe some modifications of protocol applied to rice (Oryza sativa).Seedlings grown in darkness exhibit distinct morphologies comparing with light-grown seedlings. Elongated hypocotyls, closed yellow cotyledons, and the formation of apical hooks are typical characteristics for etiolated seedlings, which are collectively named skotomorphogenesis. Various plant hormones and environmental factors are essential for maintaining skotomorphogenesis. Due to the diverse morphological outcomes in etiolated seedlings grown under different treatments, studies on skotomorphogenesis are of particular importance to reveal the molecular mechanisms underlying plant response to environmental cues. Here, we detailed experimental procedures to facilitate researchers who are investigating etiolation growth-related studies.Light is one of the most important environmental factors, serving as the energy source of photosynthesis and a cue for plant developmental programs, called photomorphogenesis. Here, we provide a standardized operation to measure physiological parameters of photomorphogenesis, including in hypocotyl length, cotyledon size, and anthocyanin content.The fundamental mechanism of light regulated plant development involves photoreceptors and their interacting proteins which act as light signaling intermediate factors. In Arabidopsis thaliana, UV RESISTANCE LOCUS 8 (UVR8) is responsible for the perception and the initiation of UV-B light signal. To data, only a few proteins have been revealed as the components of UVR8 protein complexes, limiting our understanding of the molecular mechanisms by which UV-B light input is interpreted to orchestrate numerous physiological outputs in plants. Therefore, it is necessary to isolate and identify the components of UVR8 protein complexes at a global level, in order to uncover novel UV-B light signaling factors and pathways. In this chapter, we provide a protocol for the isolation of UVR8 protein complexes. Basically, co-immunoprecipitation (co-IP) assay is employed to enrich UVR8 and its associating proteins in vivo. This method can be used coupling with specific treatments and is compatible with successive biochemical analysis.The presence of neighbor or overtopping plants is perceived by changes in light quality, which lead to several growth and developmental changes known as shade avoidance syndrome (SAS). Among them, the analysis of hypocotyl elongation is an important SAS physiological output that has been successfully used to investigate photoreceptors and downstream signaling components. Here we describe the experimental setup and growth conditions used to investigate photoreceptors and their signaling mechanisms through the analysis of hypocotyl elongation in laboratory, using simulated low R/FR ratio, low blue light, and true/deep shade conditions.Light triggers changes in plant nuclear architecture to control differentiation, adaptation, and growth. A series of genetic, molecular, and imaging approaches have revealed that the nucleus forms a hub for photo-induced protein interactions and gene regulatory events. However, the mechanism and function of light-induced nuclear compartmentalization is still unclear. This chapter provides detailed experimental protocols for examining the morphology and potential functional significance of light signaling components that localize in light-induced subnuclear domains, also known as photobodies. We describe how immunolabeling of endogenous proteins and fluorescent in situ hybridization (FISH) could be combined with confocal imaging of fluorescently tagged proteins to assess co-localization in Arabidopsis nuclei. Furthermore, we employ a super-resolution imaging approach to study the morphology of photobodies at unprecedented detail.
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