Innovation, conservation, and repurposing of gene function in root cell type development.

Plant species have evolved myriads of solutions, including complex cell type development and regulation, to adapt to dynamic environments. To understand this cellular diversity, we profiled tomato root cell type translatomes. Using xylem differentiation in tomato, examples of functional innovation, repurposing, and conservation of transcription factors are described, relative to the model plant Arabidopsis. Repurposing and innovation of genes are further observed within an exodermis regulatory network and illustrate its function. Comparative translatome analyses of rice, tomato, and Arabidopsis cell populations suggest increased expression conservation of root meristems compared with other homologous populations. In addition, the functions of constitutively expressed genes are more conserved than those of cell type/tissue-enriched genes. These observations suggest that higher order properties of cell type and pan-cell type regulation are evolutionarily conserved between plants and animals.

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.

Reprogramming of Root Cells during Nitrogen-Fixing Symbiosis Involves Dynamic Polysome Association of Coding and Noncoding RNAs.

Translational control is a widespread mechanism that allows the cell to rapidly modulate gene expression in order to provide flexibility and adaptability to eukaryotic organisms. We applied translating ribosome affinity purification combined with RNA sequencing to characterize translational regulation of mRNAs at early stages of the nitrogen-fixing symbiosis established between Medicago truncatula and Sinorhizobium meliloti Our analysis revealed a poor correlation between transcriptional and translational changes and identified hundreds of regulated protein-coding and long noncoding RNAs (lncRNAs), some of which are regulated in specific cell types. We demonstrated that a short variant of the lncRNA Trans-acting small interference RNA3 (TAS3) increased its association to the translational machinery in response to rhizobia. Functional analysis revealed that this short variant of TAS3 might act as a target mimic that captures microRNA390, contributing to reduce trans acting small interference Auxin Response Factor production and modulating nodule formation and rhizobial infection. The analysis of alternative transcript variants identified a translationally upregulated mRNA encoding subunit 3 of the SUPERKILLER complex (SKI3), which participates in mRNA decay. Knockdown of SKI3 decreased nodule initiation and development, as well as the survival of bacteria within nodules. Our results highlight the importance of translational control and mRNA decay pathways for the successful establishment of the nitrogen-fixing symbiosis.

Profiling of Accessible Chromatin Regions across Multiple Plant Species and Cell Types Reveals Common Gene Regulatory Principles and New Control Modules.

The transcriptional regulatory structure of plant genomes remains poorly defined relative to animals. It is unclear how many cis-regulatory elements exist, where these elements lie relative to promoters, and how these features are conserved across plant species. We employed the assay for transposase-accessible chromatin (ATAC-seq) in four plant species (Arabidopsis thaliana, Medicago truncatula, Solanum lycopersicum, and Oryza sativa) to delineate open chromatin regions and transcription factor (TF) binding sites across each genome. Despite 10-fold variation in intergenic space among species, the majority of open chromatin regions lie within 3 kb upstream of a transcription start site in all species. We find a common set of four TFs that appear to regulate conserved gene sets in the root tips of all four species, suggesting that TF-gene networks are generally conserved. Comparative ATAC-seq profiling of Arabidopsis root hair and non-hair cell types revealed extensive similarity as well as many cell-type-specific differences. Analyzing TF binding sites in differentially accessible regions identified a MYB-driven regulatory module unique to the hair cell, which appears to control both cell fate regulators and abiotic stress responses. Our analyses revealed common regulatory principles among species and shed light on the mechanisms producing cell-type-specific transcriptomes during development.

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.

The MicroRNA390/TAS3 Pathway Mediates Symbiotic Nodulation and Lateral Root Growth.

Legume roots form two types of postembryonic organs, lateral roots and symbiotic nodules. Nodule formation is the result of the interaction of legumes with rhizobia and requires the mitotic activation and differentiation of root cells as well as an independent, but coordinated, program that allows infection by rhizobia. MicroRNA390 (miR390) is an evolutionarily conserved microRNA that targets the Trans-Acting Short Interference RNA3 (TAS3) transcript. Cleavage of TAS3 by ARGONAUTE7 results in the production of trans-acting small interference RNAs, which target mRNAs encoding AUXIN RESPONSE FACTOR2 (ARF2), ARF3, and ARF4. Here, we show that activation of the miR390/TAS3 regulatory module by overexpression of miR390 in Medicago truncatula promotes lateral root growth but prevents nodule organogenesis, rhizobial infection, and the induction of two key nodulation genes, Nodulation Signaling Pathway1 (NSP1) and NSP2 Accordingly, inactivation of the miR390/TAS3 module, either by expression of a miR390 target mimicry construct or mutations in ARGONAUTE7, enhances nodulation and rhizobial infection, alters the spatial distribution of the nodules, and increases the percentage of nodules with multiple meristems. Our results revealed a key role of the miR390/TAS3 pathway in legumes as a modulator of lateral root organs, playing opposite roles in lateral root and nodule development.

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.

Selective recruitment of mRNAs and miRNAs to polyribosomes in response to rhizobia infection in Medicago truncatula.

Translation of mRNAs is a key regulatory step that contributes to the coordination and modulation of eukaryotic gene expression during development or adaptation to the environment. mRNA stability or translatability can be regulated by the action of small regulatory RNAs (sRNAs), which control diverse biological processes. Under low nitrogen conditions, leguminous plants associate with soil bacteria and develop a new organ specialized in nitrogen fixation: the nodule. To gain insight into the translational regulation of mRNAs during nodule formation, the association of mRNAs and sRNAs to polysomes was characterized in roots of the model legume Medicago truncatula during the symbiotic interaction with Sinorhizobium meliloti. Quantitative comparison of steady-state and polysomal mRNAs for 15 genes involved in nodulation identified a group of transcripts with slight or no change in total cellular abundance that were significantly upregulated at the level of association with polysomes in response to rhizobia. This group included mRNAs encoding receptors like kinases required either for nodule organogenesis, bacterial infection or both, and transcripts encoding GRAS and NF-Y transcription factors (TFs). Quantitative analysis of sRNAs in total and polysomal RNA samples revealed that mature microRNAs (miRNAs) were associated with the translational machinery, notably, miR169 and miR172, which target the NF-YA/HAP2 and AP2 TFs, respectively. Upon inoculation, levels of miR169 pronouncedly decreased in polysomal complexes, concomitant with the increased accumulation of the NF-YA/HAP2 protein. These results indicate that both mRNAs and miRNAs are subject to differential recruitment to polysomes, and expose the importance of selective mRNA translation during root nodule symbiosis.

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.

To keep or not to keep: mRNA stability and translatability in root nodule symbiosis.

Post-transcriptional control of gene expression allows plants to rapidly adapt to changes in their environment. Under low nitrogen conditions, legume plants engage into a symbiosis with soil bacteria that results in the formation of root nodules, where bacteria are allocated and fix atmospheric nitrogen for the plant's benefit. Recent studies highlighted the importance of small RNA-mediated mechanisms in the control of bacterial infection, nodule organogenesis, and the long-distance signaling that balances plant growth and nodulation. Examples of such mechanisms are shoot-to-root mobile microRNAs and small RNA fragments derived from degradation of bacterial transfer RNAs that repress complementary mRNAs in the host plant. Mechanisms of selective mRNA translation also contribute to rapidly modulate the expression of nodulation genes in a cell-specific manner during symbiosis. Here, the most recent advances made on the regulation of mRNA stability and translatability, and the emerging roles of long non-coding RNAs in symbiosis are summarized.

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.

Analysis of Ribosome-Associated mRNAs in Rice Reveals the Importance of Transcript Size and GC Content in Translation.

Gene expression is controlled at transcriptional and post-transcriptional levels including decoding of messenger RNA (mRNA) into polypeptides via ribosome-mediated translation. Translational regulation has been intensively studied in the model dicot plant Arabidopsis thaliana, and in this study, we assessed the translational status [proportion of steady-state mRNA associated with ribosomes] of mRNAs by Translating Ribosome Affinity Purification followed by mRNA-sequencing (TRAP-seq) in rice (Oryza sativa), a model monocot plant and the most important food crop. A survey of three tissues found that most transcribed rice genes are translated whereas few transposable elements are associated with ribosomes. Genes with short and GC-rich coding regions are overrepresented in ribosome-associated mRNAs, suggesting that the GC-richness characteristic of coding sequences in grasses may be an adaptation that favors efficient translation. Transcripts with retained introns and extended 5' untranslated regions are underrepresented on ribosomes, and rice genes belonging to different evolutionary lineages exhibited differential enrichment on the ribosomes that was associated with GC content. Genes involved in photosynthesis and stress responses are preferentially associated with ribosomes, whereas genes in epigenetic regulation pathways are the least enriched on ribosomes. Such variation is more dramatic in rice than that in Arabidopsis and is correlated with the wide variation of GC content of transcripts in rice. Taken together, variation in the translation status of individual transcripts reflects important mechanisms of gene regulation, which may have a role in evolution and diversification.

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.

Insights into post-transcriptional regulation during legume-rhizobia symbiosis.

During the past ten years, changes in the transcriptome have been assessed at different stages of the legume-rhizobia association by the use of DNA microarrays and, more recently, by RNA sequencing technologies. These studies allowed the identification of hundred or thousand of genes whose steady-state mRNA levels increase or decrease upon bacterial infection or in nodules as compared with uninfected roots. However, transcriptome based-approaches do not distinguish between mRNAs that are being actively translated, stored as messenger ribonucleoproteins (mRNPs) or targeted for degradation. Despite that the increase in steady-state levels of an mRNA does not necessarily correlate with an increase in abundance or activity of the encoded protein, this information has been commonly used to select genes that are candidates to play a role during nodule organogenesis or bacterial infection. Such criterion does not take into account the post-transcriptional mechanisms that contribute to the regulation of gene expression. One of such mechanisms, which has significant impact on gene expression, is the selective recruitment of mRNAs to the translational machinery.  Here, we review the post-transcriptional mechanisms that contribute to the regulation of gene expression in the context of the ecological and agronomical important symbiotic interaction established between roots of legumes and the nitrogen fixing bacteria collectively known as rhizobia. In addition, we discuss how the development of new technologies that allow the assessment of these regulatory layers would help to understand the genetic network governing legume rhizobia symbiosis.