Project description:Plants dynamically regulate chromatin architecture, transcription and post-transcriptional processes in accordance with developmental programs and environmental cues. Isolated nuclei can provide access to early steps in gene regulation involving chromatin as well as transcript production and processing. Here we describe transfer of the Isolation of Nuclei from TAgged specific Cell Types (INTACT) to the monocot rice (Oryza sativa L.). The purification of biotinylated nuclei was redesigned by replacing the outer nuclear envelope-targeting domain with an outer nuclear envelope-anchored domain in the Nuclear Tagging Fusion protein and codon optimization of E. coli BirA, combined in a single T-DNA construct. We also developed an inexpensive methods for INTACT, T-DNA insertion mapping, and profiling of the complete nuclear transcriptome, including a rRNA degradation procedure that minimizes pre-rRNA transcripts. The comparison of nuclear and steady-state poly(A)+ transcript populations of seedling root tips confirmed the capture of pre-mRNA and exposed distinctions between the nuclear and total RNA pool. The improved INTACT plasmid configuration for monocots and accompanying methods provide access to chromatin and pre-mRNA, paving the way for monitoring nuclear transcriptome and epigenome dynamics of specific cell-types in rice and other crop species.
Project description:Background: Single-cell reconstruction of gene regulatory programs provides an important tool to understand the cellular phenotypic variation in complex tissues and their response to endogenous and environmental stimuli. While the single-cell transcriptomes of several plant organs have been elucidated, the underlying chromatin landscapes remain largely unknown. Results: To comprehensively delineate chromatin accessibility during root development of an important crop, we applied single-cell ATAC-seq to 46,758 cells from rice root tips under normal and heat stress conditions. Our data revealed cell-type-specific accessibility variance across most of the major cell types and allowed us to identify sets of transcription factors which associate with accessible chromatin regions (ACRs). Using root hair differentiation as a model, we demonstrate that chromatin dynamics and gene expression dynamics during cell type differentiation correlate in pseudotime analyses. In addition to developmental trajectories, we describe chromatin responses to heat, and identify cell type specific accessibility changes to this key environmental stimulus. Conclusions: Our work provides a framework for the integrative analysis of regulatory dynamics in an important plant organ at single-cell resolution.
Project description:Background: Single-cell reconstruction of gene regulatory programs provides an important tool to understand the cellular phenotypic variation in complex tissues and their response to endogenous and environmental stimuli. While the single-cell transcriptomes of several plant organs have been elucidated, the underlying chromatin landscapes remain largely unknown. Results: To comprehensively delineate chromatin accessibility during root development of an important crop, we applied single-cell ATAC-seq to 46,758 cells from rice root tips under normal and heat stress conditions. Our data revealed cell-type-specific accessibility variance across most of the major cell types and allowed us to identify sets of transcription factors which associate with accessible chromatin regions (ACRs). Using root hair differentiation as a model, we demonstrate that chromatin dynamics and gene expression dynamics during cell type differentiation correlate in pseudotime analyses. In addition to developmental trajectories, we describe chromatin responses to heat, and identify cell type specific accessibility changes to this key environmental stimulus. Conclusions: Our work provides a framework for the integrative analysis of regulatory dynamics in an important plant organ at single-cell resolution.
Project description:Phosphate starvation/sufficient rice seedling, root or shoot Pi-starvation or Pi-sufficient stresses responsible rice genes, including previously unannotated genes were identified by Illumina mRNA-seq technology. 53 million reads from Pi-starvation or Pi-sufficient root or shoot tissues were uniquely mapped to the rice genome, and these included 40574 RAP3 transcripts in root and 39748 RAP3 transcripts in shoot. We compared our mRNA-seq expression data with that from Rice 44K oligomicroarray, and about 95.5% (root) and 95.4% (shoot) transcripts supported by the array were confirmed expression both by the array and by mRNA-seq, Moreover, 11888 (root) and 11098 (shoot) RAP genes which were not supported by array, were evidenced expression with mRNA-seq. Furthermore, we discovered 8590 (root) and 8193 (shoot) previously unannotated transcripts upon Pi-starvation and/or Pi-sufficient.
Project description:Climate change has increased the frequency and intensity of floods that impact global agricultural productivity. To better understand the response mechanisms and evolutionary history of gene family member regulation across angiosperm phyla, we studied the rapid submergence response of rice, the legume Medicago truncatula, and two Solanum species, domesticated tomato (S. lycopersicum cv. M82) and its dryland-adapted wild relative S. pennellii. Response to hypoxic conditions was measured by analyzing transcriptional and post-translational regulation in root tips of each species. This was achieved by the use of Nuclei Tagged in specific Cell Types (INTACT) and Translating Ribosome Affinity Purification to obtain chromatin and sub-populations of gene transcripts. (1) Chromatin accessibility was evaluated by coupling INTACT with ATAC-seq (assay for Transposon-Accessible Chromatin). (2) INTACT was used to capture nuclear RNA (nRNA). (3) Polyadenylated mRNA (polyA RNA) was obtained by standard oligo(dT) selection. (4) Ribosome-associated polyA mRNA (polyA RNA) was obtained by use of Translating Ribosome Affinity Purification (TRAP). Ribosome footprinting (Ribo-seq) was accomplished by using TRAP to capture ribosome protected fragments after RNAseI digestion. Samples evaluated include the apical root tip (four species) and shoot region (Solanum species only) under control conditions and after 2 h of submergence
Project description:Whole genome arrays have been used to analyze the transcriptomic response to vanadium stress in rice root. Identify genes and pathways that would respond to vanadium stress