Andersonjiang8086

RNA sequencing (RNA-seq) has proven invaluable for exploring gene expression variation under complex environmental cues. However, the cost of standard RNA-seq (e.g., Illumina TruSeq or NEBNext) remains a barrier for high-throughput applications. 3'-Tag RNA-seq (3'-TagSeq) is a cost-effective solution that permits large-scale experiments. Unlike standard RNA-seq, which generates sequencing libraries for full-length mRNAs, 3'-TagSeq only generates a single fragment from the 3' end of each transcript (a tag read) and quantifies gene expression by tag abundance. Consequently, 3'-TagSeq requires lower sequencing depth (~5 million reads per sample) than standard RNA-seq (~30 million reads per sample), which reduces costs and allows increased technical and biological replication in experiments. Because 3'-TagSeq is considerably cheaper than standard RNA-seq while exhibiting comparable accuracy and reproducibility, researchers focusing on gene expression levels in large or extensive time-series experiments might find 3'-TagSeq to be superior to standard RNA-seq. In this chapter, we describe 3'-TagSeq sequencing library preparation and provide example bioinformatics and statistical analyses of gene expression data.Chromatin immunoprecipitation, or ChIP, is a powerful experimental technique for probing protein-DNA interactions in vivo. This assay can be used to investigate the association of a protein of interest with specific target loci. Alternatively, it can be combined with high-throughput sequencing technology to identify genome-wide binding sites. Here, we describe a ChIP protocol that was optimized for low-abundance transcription factors in Arabidopsis, and provide guidance on how to adapt it for other types of plants and proteins.The plant circadian clock regulates multiple developmental and physiological events that occur at specific times and seasons. As many of the currently known clock proteins and clock-associated regulators are transcription factors, analyzing molecular events in the nuclei is crucial. In addition, long-time course analyses of protein abundance and interactions are often required to assess the role of the circadian clock on clock-regulated phenomena. Here we introduce a simple procedure to prepare nuclear-enriched tissues, which we routinely use to study time-resolved accumulation changes in low-abundance nuclear proteins (i.e., transcription factors). In addition to measuring changes in abundance, investigating the protein-protein interaction dynamics at specific times of day or under certain environmental conditions is needed for plant chronobiology studies. Therefore, we also present our co-immunoprecipitation method for studying diurnal/circadian protein-protein interactions, tailored to nuclear-localized proteins in Arabidopsis and tobacco.Fructans are carbohydrates present in more than 15% of flowering plants. They represent the major pool of carbohydrates in some species, especially when facing cold or drought. However, the functions of fructans with high or low degrees of polymerization (DP), their diurnal use, and the regulation of their synthesis and degradation in response to stresses still remain unclear. Here we present an enzymatic protocol adapted to 96-well microplates that simultaneously allows the determination of fructans and glucose, fructose, and sucrose. Moreover, the protocol allows to estimate the average DP of the fructans in the samples. The protocol is based on the enzymatic degradation of fructans into glucose and fructose and their subsequent conversion into gluconate 6-phosphate concomitant with the formation of NADH in the presence of ATP.Circadian clocks allow organisms to synchronize growth to occur at the most optimal time of the day. In plants, the circadian clock controls the timing of hypocotyl (seedling stem) elongation. The activity of the circadian clock subsequently results in hypocotyl elongation being restricted to a small window around dawn and the early morning. Measuring hypocotyl elongation has provided circadian biologists a quick and non-intensive experimental tool to understand the effect of a circadian mutation on plant growth. Furthermore, hypocotyl elongation is also independently regulated by light, temperature, and hormone signaling pathways. Thus, hypocotyl assays can be expanded to investigate the crosstalk between the circadian clock and other extrinsic and intrinsic signaling pathways in controlling plant development. In this chapter we describe the resources and methods required to set up and analyze hypocotyl elongation in Arabidopsis.One of the most powerful methods to identify loci controlling complex quantitative traits has been the quantitative trait locus (QTL) mapping. The QTL mapping approach has proven immensely useful to improve our understanding of key pathways such as flowering time, growth, and disease resistance. Since major circadian clock parameters such as period, phase, and amplitude are quantitative in nature, the QTL mapping approach could also be used to study the complex genetic architecture of the circadian clock. Here, we describe a simple QTL mapping method to identify components controlling clock parameters in natural populations of Arabidopsis thaliana.ODE models have been used for decades to help circadian biologists understand the rhythmic phenomena they observe and to predict the behavior of plant circadian rhythms under changed conditions such as genetic mutations or novel environments. The models vary in complexity, and for good reasons, but they share the same mathematical ingredients in their construction and the same computational methods in their solution. Here we explain the fundamental concepts which define ODE models. We sketch how ODE models can be understood, how they can be solved mathematically and computationally, and the important distinction between autonomous and non-autonomous phenomena. The concepts are illustrated with examples which illustrate the basic concepts and which may help to describe the strengths and limitations of these models and the computational investigations of their properties.Firefly luciferase is widely used as a bioluminescence reporter, which is simple, high signal-to-noise ratio and especially suitable for the long-term analysis of circadian clock-regulated gene expression. Here, we report the method of tracking circadian rhythms in Agrobacterium rhizogenes-induced soybean hairy roots via TopCount™ Microplate Scintillation Counter or Deep-Cooled CCD camera. Using transgenic soybean hairy roots, we monitored the endogenous 24-h oscillations of clock genes expression and investigated the precise parameters of circadian rhythmicity. Researchers can easily analyze the circadian phenotype in legumes and non-legumes using bioluminescence reporters carried by the hairy roots, avoiding time-consuming transgenic work.Circadian clocks are endogenous timing mechanisms that allow an organism to adapt cellular processes in anticipation of predictable changes in the environment. Luciferase reporters are well utilized as an effective, nondestructive method to measure circadian rhythms of promoter activity in Arabidopsis. Obtaining stable transgenic reporter lines can be laborious. Here, we report a protocol for Agrobacterium-mediated seedling transformation tailored for plant circadian studies. We show that period estimates generated from wild-type and clock-mutant seedlings transformed with circadian luciferase reporters are similar to rhythms obtained from equivalent stable transgenic seedlings. These experiments demonstrate the versatility and robustness of the protocol for testing new constructs or quickly assessing circadian effects in any genotype of interest.The A. thaliana circadian clock is an example of a gene network that generates rich temporal and spatial dynamics. Bioluminescent imaging has proven a powerful method to help dissect the genetic mechanisms that generate oscillations of gene expression over the course of the day. However, its use for the study of spatial regulation is often limited by resolution. Here, we describe a modified luciferase imaging method for the study of the Arabidopsis circadian clock across the plant at sub-tissue-level resolution.Monitoring prompt chlorophyll fluorescence (F) by making consecutive pulse amplitude modulation (PAM) measurements is a noninvasive, nondestructive, potentially high-throughput technique for evaluating circadian rhythms in diverse plant species. The technique is also less labor-intensive than many others currently used and requires no transgenic procedures.One of the key objectives of data analysis in circadian research is to quantify the rhythmic properties of the experimental data. BioDare2 is a free, online service which provides fast timeseries analysis, attractive visualizations, and data sharing. This chapter outlines the description of an experiment for BioDare2 and how to upload and analyze the numerical timeseries data.The circadian clock responds to light signals and therefore participates in the plant's daily response to light. The phase response curve (PRC) is typically used in the study of chronobiology to detect the effect of various environmental cues on a given circadian rhythm. In this chapter we describe protocols on measuring the setting of the light pulses at different times of a day, the measurement of circadian rhythm, and the calculation of phase shift in response to light pulses. The promoterluciferase reporter was used to provide fine rhythmic traces and the subsequent circadian parameters of mathematical analysis. A classical PRC assay to light pulses is the key experimental basis for determining the signal components of resetting the circadian clock.Previously reported in vitro release test methods for drug-releasing vaginal rings containing poorly water-soluble drugs have described use of water-alcohol systems or surfactant solutions in efforts to maintain sink conditions. Here, as part of efforts to more closely match in vitro and in vivo release for the 25 mg dapivirine matrix-type silicone elastomer vaginal ring for HIV prevention, we have investigated alternatives to the 11 v/v water/isopropanol medium described previously. Specifically, we evaluated dapivirine release from rings into (i) monophasic water/isopropanol mixtures of varying compositions and (ii) biphasic buffer/octanol systems using pH 4.2 and pH 7.0 buffers. The rate and mechanism of dapivirine release were dependent upon the isopropanol concentration in the release medium, in accordance with the observed trend in drug solubility. At 0 and 10% v/v isopropanol concentrations, dapivirine release followed a partition-controlled mechansim. For media containing ≥ 20% v/v isopropanol, in vitro release of dapivirine was significantly increased and obeyed permeation-controlled kinetics. Cumulative release of ~3.5 mg dapivirine over 28 days was obtained using a water isopropanol mixture containing 20% v/v isopropanol, similar to the ~4 mg dapivirine released in vivo. Dapivirine release into the biphasic buffer/octanol system (intended to mimic the fluid/tissue environment in vivo) was constrained by the limited solubility of dapivirine in the buffer component in which the ring resided, such that cumulative dapivirine release was consistently lower than that observed with the 20% v/v isopropanol in water medium. Release into the biphasic system was also pH dependent, in line with dapivirine's pKa and with potential implications for in vivo release and absorption in women with elevated vaginal pH.