A PXY-Mediated Transcriptional Network Integrates Signaling Mechanisms to Control Vascular Development in Arabidopsis

Vascular meristems generate the majority of biomass in higher plants. They constitute a bifacial stem cell population from which xylem and phloem are specified on opposing sides by positional signals. The PHLOEM INTERCALATED WITH XYLEM (PXY) receptor kinase promotes vascular cell division and organisation. However, how these functions are specified and integrated is unknown. Here, a putative PXY-mediated transcriptional regulatory network comprised of 690 transcription factor-promoter interactions was mapped. Among these interactions was a feed-forward loop containing transcription factors WUSCHEL HOMEOBOX RELATED 14 (WOX14) and TARGET OF MONOPTEROS 6 (TMO6), which each regulate the expression of a third transcription factor, LATERAL ORGAN BOUNDARIES DOMAIN 4 (LBD4). PXY signalling in turn regulates the WOX14, TMO6, LBD4 loop to control vascular proliferation. Genetic interaction between LBD4 and PXY suggests that LBD4 marks the phloem-procambium boundary, thus defining the shape of the vascular bundle. These data collectively support a novel mechanism that influences recruitment of cells into the phloem lineage, and defines the role of PXY signalling in this context to the arrangement of vascular tissue

Promoter-Based Integration in Plant Defense Regulation

A key unanswered question in plant biology is how a plant regulates metabolism to maximize performance across an array of biotic and abiotic environmental stresses. In this study, we addressed the potential breadth of transcriptional regulation that can alter accumulation of the defensive glucosinolate metabolites in Arabidopsis (Arabidopsis thaliana). A systematic yeast one-hybrid study was used to identify hundreds of unique potential regulatory interactions with a nearly complete complement of 21 promoters for the aliphatic glucosinolate pathway. Conducting high-throughput phenotypic validation, we showed that >75% of tested transcription factor (TF) mutants significantly altered the accumulation of the defensive glucosinolates. These glucosinolate phenotypes were conditional upon the environment and tissue type, suggesting that these TFs may allow the plant to tune its defenses to the local environment. Furthermore, the pattern of TF/promoter interactions could partially explain mutant phenotypes. This work shows that defense chemistry within Arabidopsis has a highly intricate transcriptional regulatory system that may allow for the optimization of defense metabolite accumulation across a broad array of environments

Transcriptional networks governing plant metabolism

Efficiently obtaining and utilizing energy and elements is critical for an organism to maximize its fitness. Optimizing these processes requires precise regulation and coordination of an organism's metabolic networks in response to diverse environmental conditions and developmental stages. Metabolic regulation is often considered to largely occur by allosteric feedback where the metabolites directly influence the enzymes function. Recent work is showing that there is also an extensive role for transcriptional control of the enzyme encoding genes to construct the metabolic network in response to developmental and environmental stimuli. Within this review, we go through the extensive evidence of how transcription can coordinate the necessary metabolic shifts required to coordinate a plants metabolism with its development and environment. Additionally, we discuss evidence that the metabolites not only feed-back regulate the enzymes but also the upstream transcriptional processes, possibly to stabilize the system

Preparation of Plant Material for Analysis of Protein–Nucleic Acid Interactions by FRET-FLIM

International audienceDNA-binding proteins are involved in the dynamic regulation of various cellular processes such as recombination, replication, and transcription. For investigating dynamic assembly and disassembly of molecular complexes in living cells, fluorescence microscopy represents a tremendous tool in biology. A fluorescence resonance energy transfer (FRET) approach coupled to fluorescence lifetime imaging microscopy (FLIM) has been used recently to monitor protein-DNA associations in plant cells. With this approach, the donor fluorophore is a GFP-tagged binding partner expressed in plant cells. A Sytox (R) Orange treatment converts nuclear nucleic acids to FRET acceptors. A decrease of GFP lifetime is due to FRET between donor and acceptor, indicating close association of the GFP binding partner and Sytox (R) Orange-stained DNA. In this chapter, we present a step-by-step protocol for the transient expression in N. benthamiana of GFP-tagged proteins and the fixation and permeabilization procedures used for the preparation of plant material aimed at detecting protein-nucleic acid interactions by FRET-FLIM measurements

Promoter-based integration in plant defense regulation.

A key unanswered question in plant biology is how a plant regulates metabolism to maximize performance across an array of biotic and abiotic environmental stresses. In this study, we addressed the potential breadth of transcriptional regulation that can alter accumulation of the defensive glucosinolate metabolites in Arabidopsis (Arabidopsis thaliana). A systematic yeast one-hybrid study was used to identify hundreds of unique potential regulatory interactions with a nearly complete complement of 21 promoters for the aliphatic glucosinolate pathway. Conducting high-throughput phenotypic validation, we showed that >75% of tested transcription factor (TF) mutants significantly altered the accumulation of the defensive glucosinolates. These glucosinolate phenotypes were conditional upon the environment and tissue type, suggesting that these TFs may allow the plant to tune its defenses to the local environment. Furthermore, the pattern of TF/promoter interactions could partially explain mutant phenotypes. This work shows that defense chemistry within Arabidopsis has a highly intricate transcriptional regulatory system that may allow for the optimization of defense metabolite accumulation across a broad array of environments

Promoter-Based Integration in Plant Defense Regulation

A key unanswered question in plant biology is how a plant regulates metabolism to maximize performance across an array of biotic and abiotic environmental stresses. In this study, we addressed the potential breadth of transcriptional regulation that can alter accumulation of the defensive glucosinolate metabolites in Arabidopsis (Arabidopsis thaliana). A systematic yeast one-hybrid study was used to identify hundreds of unique potential regulatory interactions with a nearly complete complement of 21 promoters for the aliphatic glucosinolate pathway. Conducting high-throughput phenotypic validation, we showed that >75% of tested transcription factor (TF) mutants significantly altered the accumulation of the defensive glucosinolates. These glucosinolate phenotypes were conditional upon the environment and tissue type, suggesting that these TFs may allow the plant to tune its defenses to the local environment. Furthermore, the pattern of TF/promoter interactions could partially explain mutant phenotypes. This work shows that defense chemistry within Arabidopsis has a highly intricate transcriptional regulatory system that may allow for the optimization of defense metabolite accumulation across a broad array of environments

Promoter-based integration in plant defense regulation.

A key unanswered question in plant biology is how a plant regulates metabolism to maximize performance across an array of biotic and abiotic environmental stresses. In this study, we addressed the potential breadth of transcriptional regulation that can alter accumulation of the defensive glucosinolate metabolites in Arabidopsis (Arabidopsis thaliana). A systematic yeast one-hybrid study was used to identify hundreds of unique potential regulatory interactions with a nearly complete complement of 21 promoters for the aliphatic glucosinolate pathway. Conducting high-throughput phenotypic validation, we showed that >75% of tested transcription factor (TF) mutants significantly altered the accumulation of the defensive glucosinolates. These glucosinolate phenotypes were conditional upon the environment and tissue type, suggesting that these TFs may allow the plant to tune its defenses to the local environment. Furthermore, the pattern of TF/promoter interactions could partially explain mutant phenotypes. This work shows that defense chemistry within Arabidopsis has a highly intricate transcriptional regulatory system that may allow for the optimization of defense metabolite accumulation across a broad array of environments

Enhanced Y1H assays for Arabidopsis

We present an Arabidopsis thaliana full-length transcription factor resource of 92% of root stele-expressed transcription factors and 74.5% of root-expressed transcription factors. We demonstrate its use with enhanced yeast one-hybrid (eY1H) screening for rapid, systematic mapping of plant transcription factor-promoter interactions. We identified 158 interactions with 13 stele-expressed promoters, many of which occur physically or are regulatory in planta