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In plants, gene expression is orchestrated by thousands of transcription factors (TFs). For instance, a large set of bHLH TFs are involved in the regulation of iron homeostasis in Arabidopsis thaliana. The identification of the direct target genes of TFs through uncovering the interaction between the TFs and cis-regulatory elements has become an essential step toward a comprehensive understanding of the iron homeostasis transcriptional regulatory network in Arabidopsis. Chromatin immunoprecipitation (ChIP) followed by qRT-PCR (ChIP-qPCR), sequencing (ChIP-seq), or chip hybridization (ChIP-chip) is a robust tool to investigate protein-DNA interactions in plants in a physiological context. The procedure generally includes six steps: DNA-protein crosslink, isolation of nuclei, shearing of chromatin, immunoprecipitation, DNA purification, and qRT-PCR analyses. In this protocol, we describe guidelines, experimental setup, and conditions for ChIP experiment in Arabidopsis. This protocol focuses on seedlings grown in control and iron deficiency conditions, but can readily be adapted for use with other Arabidopsis tissues or samples. In addition, the protocol could also be applied to perform ChIP-chip or ChIP-seq experiments., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Plants challenged with iron deficiency produce in their roots and secrete into the rhizosphere several small molecules named coumarins that derive from the phenylpropanoid pathway. Coumarins are biosynthesized in different root cell types and transported to the root epidermis prior to their secretion in the surrounding media. Taking advantage of the natural fluorescence of most coumarins glycosides when exposed to UV light, we developed a method to uncover their individual cellular localization and accumulation. This approach couples spectral imaging acquisition and linear unmixing analysis. In this protocol, we describe guidelines, experimental setup, and conditions for the analysis of coumarins localization and accumulation in Arabidopsis thaliana root seedlings grown in control and iron deficiency conditions, at both acidic and alkaline pH., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Localization and quantification of expression levels of genes help to determine their function. Localization of gene expression is often achieved through the study of their promoter activity. Three main reporter genes β-glucuronidase (GUS), green fluorescent protein (GFP), and luciferase (LUC) have been intensively used to characterize promoter activities, each having its own specificities and advantages. Among them, the LUC reporter gene is best suitable for the analysis of the promoter activity of genes in intact living plants. Here, we describe a LUC-based method that allows to precisely localize and quantify promoter activity at the whole plant level, and to study the mechanisms that are involved in long-distance regulation of gene expression in Arabidopsis thaliana. Imaging LUC signals with a low-light CCD camera allows monitoring promoter activity in time and space in the transgenic plant harboring the promoter fused with the LUC gene. In addition, it allows quantifying change of promoter activities in plant during several hours.

Transient expression assays are valuable techniques to study in vivo the transcriptional regulation of gene expression. These methods allow to assess the transcriptional properties of a given transcription factor (TF) or a complex of regulatory proteins against specific DNA motifs, called cis-regulatory elements. Here, we describe a fast, efficient, and reliable method based on the use of Physcomitrella patens protoplasts that allows the study of gene expression in a qualitative and quantitative manner by combining the advantage of GFP (green fluorescent protein) as a marker of promoter activity with flow cytometry for accurate measurement of fluorescence in individual cells.

Yeast one-hybrid (Y1H) assay has been proven to be a powerful technique to characterize in vivo the interaction between a given transcription factor (TF), or its DNA-binding domain (DBD), and target DNA sequences. Comprehensive characterization of TF/DBD and DNA interactions should allow designing synthetic promoters that would undoubtedly be valuable for biotechnological approaches. Here, we use the ligation-independent cloning system (LIC) in order to enhance the cloning efficiency of DNA motifs into the pHISi Y1H vector. LIC overcomes important limitations of traditional cloning technologies, since any DNA fragment can be cloned into LIC compatible vectors without using restriction endonucleases, ligation, or in vitro recombination.