The Arabidopsis transcription factor NLP2 regulates early nitrate responses and integrates nitrate assimilation with energy and carbon skeleton supply.

Nitrate signaling improves plant growth under limited nitrate availability and, hence, optimal resource use for crop production. Whereas several transcriptional regulators of nitrate signaling have been identified, including the Arabidopsis thaliana transcription factor NIN-LIKE PROTEIN7 (NLP7), additional regulators are expected to fine-tune this pivotal physiological response. Here, we characterized Arabidopsis NLP2 as a top-tier transcriptional regulator of the early nitrate response gene regulatory network. NLP2 interacts with NLP7 in vivo and shares key molecular features such as nitrate-dependent nuclear localization, DNA-binding motif, and some target genes with NLP7. Genetic, genomic, and metabolic approaches revealed a specific role for NLP2 in the nitrate-dependent regulation of carbon and energy-related processes that likely influence plant growth under distinct nitrogen environments. Our findings highlight the complementarity and specificity of NLP2 and NLP7 in orchestrating a multitiered nitrate regulatory network that links nitrate assimilation with carbon and energy metabolism for efficient nitrogen use and biomass production.

Custom methods to identify conserved genetic modules applied to novel transcriptomic data from Amborella trichopoda.

We have devised a procedure for the inter-species comparison of transcriptomic data and used this procedure to reconstruct the expression dynamics of major genetic modules that were present at least 149 million years ago in the most recent common ancestor of living angiosperms. We began by using laser-assisted microdissection to generate novel transcriptomic data from female flower tissues of Amborella trichopoda, the likely sister to all other living angiosperms. We then employed a gene-expression clustering method, followed by a custom procedure to compare genetic modules on the basis of gene orthology between Amborella and the molecular-genetic model angiosperm Arabidopsis thaliana. Using this protocol, we succeeded in identifying nine major genetic modules that appear to have conserved their expression dynamics from an early stage in angiosperm evolution. The genes of these modules, representing over 5000 orthogroups, include around one third of those known to control female reproductive development in Arabidopsis. Our study constitutes a proof of concept for the comparison of transcriptomic data between widely diverged plant species and represents a first step in the large-scale analysis of gene expression dynamics in a macro-evolutionary context.

Retrograde signalling from the mitochondria to the nucleus translates the positive effect of ethylene on dormancy breaking of Arabidopsis thaliana seeds.

Ethylene and reactive oxygen species (ROS) regulate seed dormancy alleviation, but the molecular basis of their action and crosstalk remains largely unknown. Here we studied the mechanism of Arabidopsis seed dormancy release by ethylene using cell imaging, and genetic and transcriptomics approaches, in order to tackle its possible interaction with ROS homeostasis. We found that the effect of ethylene on seed germination required ROS production by the mitochondrial electron transport chain. Seed response to ethylene involved a mitochondrial retrograde response (MRR) through nuclear ROS production and upregulation of the MRR components AOX1a and ANAC013, but also required the activation of the ethylene canonical pathway. Together our data allowed deciphering of the mode of action of ethylene on seed germination and the associated dynamics of ROS production. Our findings highlight the occurrence of retrograde signalling in seed germination.

Outgrowth of the axillary bud in rose is controlled by sugar metabolism and signalling.

Shoot branching is a pivotal process during plant growth and development, and is antagonistically orchestrated by auxin and sugars. In contrast to extensive investigations on hormonal regulatory networks, our current knowledge on the role of sugar signalling pathways in bud outgrowth is scarce. Based on a comprehensive stepwise strategy, we investigated the role of glycolysis/the tricarboxylic acid (TCA) cycle and the oxidative pentose phosphate pathway (OPPP) in the control of bud outgrowth. We demonstrated that these pathways are necessary for bud outgrowth promotion upon plant decapitation and in response to sugar availability. They are also targets of the antagonistic crosstalk between auxin and sugar availability. The two pathways act synergistically to down-regulate the expression of BRC1, a conserved inhibitor of shoot branching. Using Rosa calluses stably transformed with GFP-fused promoter sequences of RhBRC1 (pRhBRC1), glycolysis/TCA cycle and the OPPP were found to repress the transcriptional activity of pRhBRC1 cooperatively. Glycolysis/TCA cycle- and OPPP-dependent regulations involve the -1973/-1611 bp and -1206/-709 bp regions of pRhBRC1, respectively. Our findings indicate that glycolysis/TCA cycle and the OPPP are integrative parts of shoot branching control and can link endogenous factors to the developmental programme of bud outgrowth, likely through two distinct mechanisms.

In situ transcriptomic and metabolomic study of the loss of photosynthesis in the leaves of mixotrophic plants exploiting fungi.

Mycoheterotrophic plants have lost photosynthesis and obtain carbon through mycorrhizal fungi colonizing their roots. They are likely to have evolved from mixotrophic ancestors, which rely on both photosynthesis and fungal carbon for their development. Whereas our understanding of the ecological and genomic changes associated with the evolutionary shift to mycoheterotrophy is deepening, little information is known about the specific metabolic and physiological features driving this evolution. We investigated this issue in naturally occurring achlorophyllous variants of temperate mixotrophic orchids. We carried out an integrated transcriptomic and metabolomic analysis of the response to achlorophylly in the leaves of three mixotrophic species sampled in natura. Achlorophyllous leaves showed major impairment of their photosynthetic and mineral nutrition functions, strong accumulation of free amino acids, overexpression of enzymes and transporters related to sugars, amino acids and fatty acid catabolism, as well as induction of some autophagy-related and biotic stress genes. Such changes were reminiscent of these reported for variegated leaves and appeared to be symptomatic of a carbon starvation response. Rather than decisive metabolic innovations, we suggest that the evolution towards mycoheterotrophy in orchids is more likely to be reliant on the versatility of plant metabolism and an ability to exploit fungal organic resources, especially amino acids, to replace missing photosynthates.

Integrating multiple omics to identify common and specific molecular changes occurring in Arabidopsis under chronic nitrate and sulfate limitations.

Plants have fundamental dependences on nitrogen and sulfur and frequently have to cope with chronic limitations when their supply is sub-optimal. This study aimed at characterizing the metabolomic, proteomic, and transcriptomic changes occurring in Arabidopsis leaves under chronic nitrate (Low-N) and chronic sulfate (Low-S) limitations in order to compare their effects, determine interconnections, and examine strategies of adaptation. Metabolite profiling globally revealed opposite effects of Low-S and Low-N on carbohydrate and amino acid accumulations, whilst proteomic data showed that both treatments resulted in increases in catabolic processes, stimulation of mitochondrial and cytosolic metabolism, and decreases in chloroplast metabolism. Lower abundances of ribosomal proteins and translation factors under Low-N and Low-S corresponded with growth limitation. At the transcript level, the major and specific effect of Low-N was the enhancement of expression of defence and immunity genes. The main effect of chronic Low-S was a decrease in transcripts of genes involved in cell division, DNA replication, and cytoskeleton, and an increase in the expression of autophagy genes. This was consistent with a role of target-of-rapamycin kinase in the control of plant metabolism and cell growth and division under chronic Low-S. In addition, Low-S decreased the expression of several NLP transcription factors, which are master actors in nitrate sensing. Finally, both the transcriptome and proteome data indicated that Low-S repressed glucosinolate synthesis, and that Low-N exacerbated glucosinolate degradation. This showed the importance of glucosinolate as buffering molecules for N and S management.

Two interacting PPR proteins are major Arabidopsis editing factors in plastid and mitochondria.

RNA editing is converting hundreds of cytosines into uridines during organelle gene expression of land plants. The pentatricopeptide repeat (PPR) proteins are at the core of this posttranscriptional RNA modification. Even if a PPR protein defines the editing site, a DYW domain of the same or another PPR protein is believed to catalyze the deamination. To give insight into the organelle RNA editosome, we performed tandem affinity purification of the plastidial CHLOROPLAST BIOGENESIS 19 (CLB19) PPR editing factor. Two PPR proteins, dually targeted to mitochondria and chloroplasts, were identified as potential partners of CLB19. These two proteins, a P-type PPR and a member of a small PPR-DYW subfamily, were shown to interact in yeast. Insertional mutations resulted in embryo lethality that could be rescued by embryo-specific complementation. A transcriptome analysis of these complemented plants showed major editing defects in both organelles with a very high PPR type specificity, indicating that the two proteins are core members of E+-type PPR editosomes.

Two interacting proteins are necessary for the editing of the NdhD-1 site in Arabidopsis plastids.

After transcription, mRNA editing in angiosperm chloroplasts and mitochondria results in the conversion of cytidine to uridine by deamination. Analysis of Arabidopsis thaliana mutants affected in RNA editing have shown that many pentatricopeptide repeat proteins (PPRs) are required for specific cytidine deamination events. PPR proteins have been shown to be sequence-specific RNA binding proteins allowing the recognition of the C to be edited. The C-terminal DYW domain present in many editing factors has been proposed to catalyze C deamination, as it shows sequence similarities with cytidine deaminases in other organisms. However, many editing factors, such as the first to be discovered, CHLORORESPIRATORY REDUCTION4 (CRR4), lack this domain, so its importance has been unclear. Using a reverse genetic approach, we identified DYW1, an RNA editing factor acting specifically on the plastid ndhD-1 editing site recognized by CRR4. Unlike other known editing factors, DYW1 contains no identifiable PPR motifs but does contain a clear DYW domain. We were able to show interaction between CRR4 and DYW1 by bimolecular fluorescence complementation and to reconstitute a functional chimeric CRR4-DYW1 protein complementing the crr4 dyw1double mutant. We propose that CRR4 and DYW1 act together to edit the ndhD-1 site.

The cytidine deaminase signature HxE(x)n CxxC of DYW1 binds zinc and is necessary for RNA editing of ndhD-1.

In flowering plants, RNA editing involves deamination of specific cytidines to uridines in both mitochondrial and chloroplast transcripts. Pentatricopeptide repeat (PPR) proteins and multiple organellar RNA editing factor (MORF) proteins have been shown to be involved in RNA editing but none have been shown to possess cytidine deaminase activity. The DYW domain of some PPR proteins contains a highly conserved signature resembling the zinc-binding active site motif of known nucleotide deaminases. We modified these highly conserved amino acids in the DYW motif of DYW1, an editing factor required for editing of the ndhD-1 site in Arabidopsis chloroplasts. We demonstrate that several amino acids of this signature motif are required for RNA editing in vivo and for zinc binding in vitro. We conclude that the DYW domain of DYW1 has features in common with cytidine deaminases, reinforcing the hypothesis that this domain forms part of the active enzyme that carries out RNA editing in plants.

The Use of Nanopore Sequencing to Analyze the Chloroplast Transcriptome Part I: Library Preparation.

Global understanding of plastid gene expression has always been impaired by its complexity. RNA splicing, editing, and intercistronic processing create multiple transcripts isoforms that can hardly be resolved using traditional molecular biology techniques. During the last decade, the wide adoption of RNA-seq-based techniques has, however, allowed an unprecedented understanding of all the different steps of chloroplast gene expression, from transcription to translation. Current strategies are nonetheless unable to identify and quantify full length transcripts isoforms, a limitation that can now be overcome using Nanopore Sequencing. We here provide a complete protocol to produce, from total leaf RNA, cDNA libraries ready for Nanopore sequencing. While most Nanopore protocols take advantage of the mRNA polyA tail we here first ligate an RNA adapter to the 3' ends of the RNAs and use it to initiate the template switching reverse transcription. The cDNA is then prepared and indexed for use with the regular Oxford Nanopore v14 chemistry. This protocol is of particular interest to researchers willing to simultaneously study the multiple post-transcriptional processes prevalent in the chloroplast.

DiffSegR: an RNA-seq data driven method for differential expression analysis using changepoint detection.

To fully understand gene regulation, it is necessary to have a thorough understanding of both the transcriptome and the enzymatic and RNA-binding activities that shape it. While many RNA-Seq-based tools have been developed to analyze the transcriptome, most only consider the abundance of sequencing reads along annotated patterns (such as genes). These annotations are typically incomplete, leading to errors in the differential expression analysis. To address this issue, we present DiffSegR - an R package that enables the discovery of transcriptome-wide expression differences between two biological conditions using RNA-Seq data. DiffSegR does not require prior annotation and uses a multiple changepoints detection algorithm to identify the boundaries of differentially expressed regions in the per-base log2 fold change. In a few minutes of computation, DiffSegR could rightfully predict the role of chloroplast ribonuclease Mini-III in rRNA maturation and chloroplast ribonuclease PNPase in (3'/5')-degradation of rRNA, mRNA and tRNA precursors as well as intron accumulation. We believe DiffSegR will benefit biologists working on transcriptomics as it allows access to information from a layer of the transcriptome overlooked by the classical differential expression analysis pipelines widely used today. DiffSegR is available at https://aliehrmann.github.io/DiffSegR/index.html.

Impact of high atmospheric carbon dioxide on the biotic stress response of the model cereal species Brachypodium distachyon.

Losses due to disease and climate change are among the most important issues currently facing crop production. It is therefore important to establish the impact of climate change, and particularly of high carbon dioxide (hCO2), on plant immunity in cereals, which provide 60% of human calories. The aim of this study was to determine if hCO2 impacts Brachypodium distachyon immunity, a model plant for temperate cereals. Plants were grown in air (430 ppm CO2) and at two high CO2 conditions, one that is relevant to projections within the coming century (1000 ppm) and a concentration sufficient to saturate photosynthesis (3000 ppm). The following measurements were performed: phenotyping and growth, salicylic acid contents, pathogen resistance tests, and RNAseq analysis of the transcriptome. Improved shoot development was observed at both 1000 and 3000 ppm. A transcriptomic analysis pointed to an increase in primary metabolism capacity under hCO2. Alongside this effect, up-regulation of genes associated with secondary metabolism was also observed. This effect was especially evident for the terpenoid and phenylpropanoid pathways, and was accompanied by enhanced expression of immunity-related genes and accumulation of salicylic acid. Pathogen tests using the fungus Magnaporthe oryzae revealed that hCO2 had a complex effect, with enhanced susceptibility to infection but no increase in fungal development. The study reveals that immunity in B. distachyon is modulated by growth at hCO2 and allows identification of pathways that might play a role in this effect.

An mTRAN-mRNA interaction mediates mitochondrial translation initiation in plants.

Plant mitochondria represent the largest group of respiring organelles on the planet. Plant mitochondrial messenger RNAs (mRNAs) lack Shine-Dalgarno-like ribosome-binding sites, so it is unknown how plant mitoribosomes recognize mRNA. We show that "mitochondrial translation factors" mTRAN1 and mTRAN2 are land plant-specific proteins, required for normal mitochondrial respiration chain biogenesis. Our studies suggest that mTRANs are noncanonical pentatricopeptide repeat (PPR)-like RNA binding proteins of the mitoribosomal "small" subunit. We identified conserved Adenosine (A)/Uridine (U)-rich motifs in the 5' regions of plant mitochondrial mRNAs. mTRAN1 binds this motif, suggesting that it is a mitoribosome homing factor to identify mRNAs. We demonstrate that mTRANs are likely required for translation of all plant mitochondrial mRNAs. Plant mitochondrial translation initiation thus appears to use a protein-mRNA interaction that is divergent from bacteria or mammalian mitochondria.

Antagonistic Effect of Sucrose Availability and Auxin on Rosa Axillary Bud Metabolism and Signaling, Based on the Transcriptomics and Metabolomics Analysis.

Shoot branching is crucial for successful plant development and plant response to environmental factors. Extensive investigations have revealed the involvement of an intricate regulatory network including hormones and sugars. Recent studies have demonstrated that two major systemic regulators-auxin and sugar-antagonistically regulate plant branching. However, little is known regarding the molecular mechanisms involved in this crosstalk. We carried out two complementary untargeted approaches-RNA-seq and metabolomics-on explant stem buds fed with different concentrations of auxin and sucrose resulting in dormant and non-dormant buds. Buds responded to the combined effect of auxin and sugar by massive reprogramming of the transcriptome and metabolome. The antagonistic effect of sucrose and auxin targeted several important physiological processes, including sink strength, the amino acid metabolism, the sulfate metabolism, ribosome biogenesis, the nucleic acid metabolism, and phytohormone signaling. Further experiments revealed a role of the TOR-kinase signaling pathway in bud outgrowth through at least downregulation of Rosa hybrida BRANCHED1 (RhBRC1). These new findings represent a cornerstone to further investigate the diverse molecular mechanisms that drive the integration of endogenous factors during shoot branching.

The Analysis of the Editing Defects in the dyw2 Mutant Provides New Clues for the Prediction of RNA Targets of Arabidopsis E+-Class PPR Proteins.

C to U editing is one of the post-transcriptional steps which are required for the proper expression of chloroplast and mitochondrial genes in plants. It depends on several proteins acting together which include the PLS-class pentatricopeptide repeat proteins (PPR). DYW2 was recently shown to be required for the editing of many sites in both organelles. In particular almost all the sites associated with the E+ subfamily of PPR proteins are depending on DYW2, suggesting that DYW2 is required for the function of E+-type PPR proteins. Here we strengthened this link by identifying 16 major editing sites controlled by 3 PPR proteins: OTP90, a DYW-type PPR and PGN and MEF37, 2 E+-type PPR proteins. A re-analysis of the DYW2 editotype showed that the 49 sites known to be associated with the 18 characterized E+-type PPR proteins all depend on DYW2. Considering only the 288 DYW2-dependent editing sites as potential E+-type PPR sites, instead of the 795 known editing sites, improves the performances of binding predictions systems based on the PPR code for E+-type PPR proteins. However, it does not compensate for poor binding predictions.

Analysis of the DNA-Binding Activities of the Arabidopsis R2R3-MYB Transcription Factor Family by One-Hybrid Experiments in Yeast.

The control of growth and development of all living organisms is a complex and dynamic process that requires the harmonious expression of numerous genes. Gene expression is mainly controlled by the activity of sequence-specific DNA binding proteins called transcription factors (TFs). Amongst the various classes of eukaryotic TFs, the MYB superfamily is one of the largest and most diverse, and it has considerably expanded in the plant kingdom. R2R3-MYBs have been extensively studied over the last 15 years. However, DNA-binding specificity has been characterized for only a small subset of these proteins. Therefore, one of the remaining challenges is the exhaustive characterization of the DNA-binding specificity of all R2R3-MYB proteins. In this study, we have developed a library of Arabidopsis thaliana R2R3-MYB open reading frames, whose DNA-binding activities were assayed in vivo (yeast one-hybrid experiments) with a pool of selected cis-regulatory elements. Altogether 1904 interactions were assayed leading to the discovery of specific patterns of interactions between the various R2R3-MYB subgroups and their DNA target sequences and to the identification of key features that govern these interactions. The present work provides a comprehensive in vivo analysis of R2R3-MYB binding activities that should help in predicting new DNA motifs and identifying new putative target genes for each member of this very large family of TFs. In a broader perspective, the generated data will help to better understand how TF interact with their target DNA sequences.

Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications.

Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics.