Nuclear and cytoplasmic lncRNAs in root tips of the model legume Medicago truncatula under control and submergence.

In this study, we aimed to identify long noncoding RNAs (lncRNAs) in root tips of the model legume Medicago truncatula using previously generated nuclear, total polyA, ribosome-associated polyA, and Riboseq RNA datasets, which might shed light on their localization and potential regulatory roles. RNA-seq data were mapped to the version 5 of the M. truncatula A17 genome and analyzed to identify genome annotated lncRNAs and putative new root tip (NRT) lncRNAs. lncRNAs were classified according to their genomic location relative to chromatin accessible regions, protein-coding genes and transposable elements (TE), finding differences between annotated lncRNAs and NRT lncRNAs, both in their genomic position as well as in the type of TEs in their vicinity. We investigated their response to submergence and found a set of regulated lncRNAs that were preferentially upregulated in the nucleus, some of which were located nearby genes of the conserved submergence upregulated gene families, and chromatin accessible regions suggesting a potential regulatory role. Finally, the accumulation of lncRNAs under submergence was validated by reverse transcription quantitative polymerase chain reaction on nuclear RNA, providing additional evidence of their localization, which could ultimately be required for their function.

Nuclear Transcriptomes at High Resolution Using Retooled INTACT.

Isolated nuclei 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 of the nuclear tagging fusion (NTF) protein with an outer nuclear-envelope-anchored domain. This modified NTF was combined with codon-optimized Escherichia coli BirA in a single T-DNA construct. We also developed inexpensive methods for INTACT, T-DNA insertion mapping, and profiling of the complete nuclear transcriptome, including a ribosomal RNA degradation procedure that minimizes pre-ribosomal RNA (pre-rRNA) transcripts. A high-resolution comparison of nuclear and steady-state poly(A)+ transcript populations of seedling root tips confirmed the capture of pre-messenger RNA (pre-mRNA) and exposed distinctions in diversity and abundance of the nuclear and total transcriptomes. This retooled INTACT can enable high-resolution monitoring of the nuclear transcriptome and chromatin in specific cell types of rice and other species.

Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model.

Agrobacterium rhizogenes (or Rhizobium rhizogenes) is able to transform plant genomes and induce the production of hairy roots. We describe the use of A. rhizogenes in tomato (Solanum spp.) to rapidly assess gene expression and function. Gene expression of reporters is indistinguishable in plants transformed by Agrobacterium tumefaciens as compared with A. rhizogenes. A root cell type- and tissue-specific promoter resource has been generated for domesticated and wild tomato (Solanum lycopersicum and Solanum pennellii, respectively) using these approaches. Imaging of tomato roots using A. rhizogenes coupled with laser scanning confocal microscopy is facilitated by the use of a membrane-tagged protein fused to a red fluorescent protein marker present in binary vectors. Tomato-optimized isolation of nuclei tagged in specific cell types and translating ribosome affinity purification binary vectors were generated and used to monitor associated messenger RNA abundance or chromatin modification. Finally, transcriptional reporters, translational reporters, and clustered regularly interspaced short palindromic repeats-associated nuclease9 genome editing demonstrate that SHORT-ROOT and SCARECROW gene function is conserved between Arabidopsis (Arabidopsis thaliana) and tomato.

Gene regulatory networks shape developmental plasticity of root cell types under water extremes in rice.

Understanding how roots modulate development under varied irrigation or rainfall is crucial for development of climate-resilient crops. We established a toolbox of tagged rice lines to profile translating mRNAs and chromatin accessibility within specific cell populations. We used these to study roots in a range of environments: plates in the lab, controlled greenhouse stress and recovery conditions, and outdoors in a paddy. Integration of chromatin and mRNA data resolves regulatory networks of the following: cycle genes in proliferating cells that attenuate DNA synthesis under submergence; genes involved in auxin signaling, the circadian clock, and small RNA regulation in ground tissue; and suberin biosynthesis, iron transporters, and nitrogen assimilation in endodermal/exodermal cells modulated with water availability. By applying a systems approach, we identify known and candidate driver transcription factors of water-deficit responses and xylem development plasticity. Collectively, this resource will facilitate genetic improvements in root systems for optimal climate resilience.

TRAP-SEQ of Eukaryotic Translatomes Applied to the Detection of Polysome-Associated Long Noncoding RNAs.

Translating ribosome affinity purification (TRAP) technology allows the isolation of polysomal complexes and the RNAs associated with at least one 80S ribosome. TRAP consists of the stabilization and affinity purification of polysomes containing a tagged version of a ribosomal protein. Quantitative assessment of the TRAP RNA is achieved by direct sequencing (TRAP-SEQ), which provides accurate quantitation of ribosome-associated RNAs, including long noncoding RNAs (lncRNAs). Here we present an updated procedure for TRAP-SEQ, as well as a primary analysis guide for identification of ribosome-associated lncRNAs. This methodology enables the study of dynamic association of lncRNAs by assessing rapid changes in their transcript levels in polysomes at organ or cell-type level, during development, or in response to endogenous or exogenous stimuli.

Evolutionary flexibility in flooding response circuitry in angiosperms.

Flooding due to extreme weather threatens crops and ecosystems. To understand variation in gene regulatory networks activated by submergence, we conducted a high-resolution analysis of chromatin accessibility and gene expression at three scales of transcript control in four angiosperms, ranging from a dryland-adapted wild species to a wetland crop. The data define a cohort of conserved submergence-activated genes with signatures of overlapping cis regulation by four transcription factor families. Syntenic genes are more highly expressed than nonsyntenic genes, yet both can have the cis motifs and chromatin accessibility associated with submergence up-regulation. Whereas the flexible circuitry spans the eudicot-monocot divide, the frequency of specific cis motifs, extent of chromatin accessibility, and degree of submergence activation are more prevalent in the wetland crop and may have adaptive importance.

Isolation of Nuclei in Tagged Cell Types (INTACT), RNA Extraction and Ribosomal RNA Degradation to Prepare Material for RNA-Seq.

Gene expression is dynamically regulated on many levels, including chromatin accessibility and transcription. In order to study these nuclear regulatory events, we describe our method to purify nuclei with Isolation of Nuclei in TAgged Cell Types (INTACT). As nuclear RNA is low in polyadenylated transcripts and conventional pulldown methods would not capture non-polyadenylated pre-mRNA, we also present our method to remove ribosomal RNA from the total nuclear RNA in preparation for nuclear RNA-Seq.

Translating Ribosome Affinity Purification (TRAP) followed by RNA sequencing technology (TRAP-SEQ) for quantitative assessment of plant translatomes.

Translating Ribosome Affinity Purification (TRAP) is a technology to isolate the population of mRNAs associated with at least one 80S ribosome, referred as the translatome. TRAP is based on the expression of an epitope-tagged version of a ribosomal protein and the affinity purification of ribosomes and associated mRNAs using antibodies conjugated to agarose beads. Quantitative assessment of the translatome is achieved by direct RNA sequencing (RNA-SEQ), which provides accurate quantitation of ribosome-associated mRNAs and reveals alternatively spliced isoforms. Here we present a detailed procedure for TRAP, as well as a guide for preparation of RNA-SEQ libraries (TRAP-SEQ) and a primary data analysis. This methodology enables the study of translational dynamic by assessing rapid changes in translatomes, at organ or cell-type level, during development or in response to endogenous or exogenous stimuli.