Plants display numerous strategies to cope with phosphate (Pi)-deficiency. Despite multiple genetic studies, the molecular mechanisms of low-Pi-signalling remain unknown. To validate the interest of chemical genetics to investigate this pathway we discovered and analysed the effects of PHOSTIN (PSN), a drug mimicking Pi-starvation in Arabidopsis. We assessed the effects of PSN and structural analogues on the induction of Pi-deficiency responses in mutants and wild-type and followed their accumulation in plants organs by high pressure liquid chromotography (HPLC) or mass-spectrophotometry. We show that PSN is cleaved in the growth medium, releasing its active motif (PSN11), which accumulates in plants roots. Despite the overaccumulation of Pi in the roots of treated plants, PSN11 elicits both local and systemic Pi-starvation effects. Nevertheless, albeit that the transcriptional activation of low-Pi genes by PSN11 is lost in the phr1;phl1 double mutant, neither PHO1 nor PHO2 are required for PSN11 effects. The range of local and systemic responses to Pi-starvation elicited, and their dependence on the PHR1/PHL1 function suggests that PSN11 affects an important and early step of Pi-starvation signalling. Its independence from PHO1 and PHO2 suggest the existence of unknown pathway(s), showing the usefulness of PSN and chemical genetics to bring new elements to this field.

Arabidopsis ESD7 locus encodes the catalytic subunit of the DNA Pol ϵ involved in the synthesis of the DNA leading strand and is essential for embryo viability. The hypomorphic allele esd7-1 is viable but displays a number of pleiotropic phenotypic alterations including an acceleration of flowering time. Furthermore, Pol ϵ is involved in the epigenetic silencing of the floral integrator genes FT and SOC1, but the molecular nature of the transcriptional gene silencing mechanisms involved remains elusive. Here we reveal that ESD7 interacts with components of the PRC2 such as CLF, EMF2 and MSI1, and that mutations in ESD7 cause a decrease in the levels of the H3K27me3 mark present in the chromatin of FT and SOC1 We also demonstrate that a domain of the C-terminal region of ESD7 mediates the binding to the different PRC2 components and this interaction is necessary for the proper recruitment of PRC2 to FT and SOC1 chromatin. We unveil the existence of interplay between the DNA replication machinery and the PcG complexes in epigenetic transcriptional silencing. These observations provide an insight into the mechanisms ensuring that the epigenetic code at pivotal loci in developmental control is faithfully transmitted to the progeny of eukaryotic cells.

Deposition of the H2A.Z histone variant by the SWR1 complex (SWR1-C) in regulatory regions of specific loci modulates transcription. Characterization of mutations in Arabidopsis thaliana homologs of yeast SWR1-C has revealed a role for H2A.Z exchange in a variety of developmental processes. Nevertheless, the exact composition of plant SWR1-C and how it is recruited to target genes remains to be established. Here we show that SWC4, the Arabidopsis homolog of yeast SANT domain protein Swc4/Eaf2, is a DNA-binding protein that interacts with SWR1-C subunits. We demonstrate that the swc4-1 knockout mutant is embryo-lethal, while SWC4 RNAi knockdown lines display pleiotropic phenotypic alterations in vegetative and reproductive traits, including acceleration of flowering time, indicating that SWC4 controls post-embryonic processes. Transcriptomic analyses and genome-wide profiling of H2A.Z indicate that SWC4 represses transcription of a number of genes, including the floral integrator FT and key transcription factors, mainly by modulating H2A.Z deposition. Interestingly, SWC4 silencing does not affect H2A.Z deposition at the FLC locus nor expression of this gene, a master regulator of flowering previously shown to be controlled by SWR1-C. Importantly, we find that SWC4 recognizes specific AT-rich DNA elements in the chromatin regions of target genes and that SWC4 silencing impairs SWR1-C binding at FT. Collectively, our data suggest that SWC4 regulates plant growth and development by aiding SWR1-C recruitment and modulating H2A.Z deposition.

Environmental cues profoundly modulate cell proliferation and cell elongation to inform and direct plant growth and development. External phosphate (Pi) limitation inhibits primary root growth in many plant species. However, the underlying Pi sensory mechanisms are unknown. Here we genetically uncouple two Pi sensing pathways in the root apex of Arabidopsis thaliana. First, the rapid inhibition of cell elongation in the transition zone is controlled by transcription factor STOP1, by its direct target, ALMT1, encoding a malate channel, and by ferroxidase LPR1, which together mediate Fe and peroxidase-dependent cell wall stiffening. Second, during the subsequent slow inhibition of cell proliferation in the apical meristem, which is mediated by LPR1-dependent, but largely STOP1-ALMT1-independent, Fe and callose accumulate in the stem cell niche, leading to meristem reduction. Our work uncovers STOP1 and ALMT1 as a signalling pathway of low Pi availability and exuded malate as an unexpected apoplastic inhibitor of root cell wall expansion.

Programmed cell death (PCD) is essential for several aspects of plant life. We previously identified the mips1 mutant of Arabidopsis thaliana, which is deficient for the enzyme catalysing myo-inositol synthesis, and that displays light-dependent formation of lesions on leaves due to Salicylic Acid (SA) over-accumulation. Rationale of this work was to identify novel regulators of plant PCD using a genetic approach. A screen for secondary mutations that abolish the mips1 PCD phenotype identified a mutation in the BIG gene, encoding a factor of unknown molecular function that was previously shown to play pleiotropic roles in plant development and defence. Physiological analyses showed that BIG is required for lesion formation in mips1 via SA-dependant signalling. big mutations partly rescued transcriptomic and metabolomics perturbations as stress-related phytohormones homeostasis. In addition, since loss of function of the ceramide synthase LOH2 was not able to abolish cell death induction in mips1, we show that PCD induction is not fully dependent of sphingolipid accumulation as previously suggested. Our results provide further insights into the role of the BIG protein in the control of MIPS1-dependent cell death and also into the impact of sphingolipid homeostasis in this pathway.

Thymidine kinase 1 (TK1) phosphorylates thymidine nucleosides to generate thymidine monophosphate. This reaction belongs to the pyrimidine salvage route that is phylogenetically conserved. In the model plant Arabidopsis thaliana, TK activity contributes to maintain nuclear and organellar genome integrity by providing deoxythymidine-triphosphate (dTTP) for DNA synthesis. Arabidopsis has two TK1 genes (TK1a and TK1b) and double mutants show an albino phenotype and develop poorly. In contrast, maize (Zea mays L.) has a single TK1 (ZmTK1) gene and mutant plants are albino and display reduced genome copy number in chloroplasts. We studied the role of ZmTK1 during development and genotoxic stress response by assessing its activity at different developmental stages and by complementing Arabidopsis tk1 mutants. We found that ZmTK1 transcripts and activity are present during germination and throughout maize development. We show that ZmTK1 translocation to chloroplasts depends on a 72-amino-acid N-signal and its plastid localization is consistent with its ability to complement Arabidopsis tk1b mutants which are hypersensitive to ciprofloxacin (CIP), a genotoxic agent to organellar DNA. Also, ZmTK1 partly complemented the Arabidopsis double mutant plants during development. Our results contribute to the understanding of TK1 function in monocot species as an organellar enzyme for genome replication and repair.

Even though petals are homoplastic structures, their identity consistently involves genes of the APETALA3 (AP3) lineage. However, the extent to which the networks downstream of AP3 are conserved in species with petals of different evolutionary origins is unknown. In Ranunculaceae, the specificity of the AP3-III lineage offers a great opportunity to identify the petal gene regulatory network in a comparative framework. Using a transcriptomic approach, we investigated putative target genes of the AP3-III ortholog NdAP3-3 in Nigella damascena at early developmental stages when petal identity is determined, and we compared our data with that from selected eudicot species. We generated a de novo reference transcriptome to carry out a differential gene expression analysis between the wild-type and mutant NdAP3-3 genotypes differing by the presence vs. absence of petals at early stages of floral development. Among the 1,620 genes that were significantly differentially expressed between the two genotypes, functional annotation suggested a large involvement of nuclear activities, including regulation of transcription, and enrichment in processes linked to cell proliferation. Comparing with Arabidopsis data, we found that highly conserved genes between the two species are enriched in homologs of direct targets of the AtAP3 protein. Integrating AP3-3 binding site data from another Ranunculaceae species, Aquilegia coerulea, allowed us to identify a set of 18 putative target genes that were conserved between the three species. Our results suggest that, despite the independent evolutionary origin of petals in core eudicots and Ranunculaceae, a small conserved set of genes determines petal identity and early development in these taxa.

Elevated growth temperatures are negatively affecting crop productivity by increasing yield losses. The modulation of root traits associated with improved response to rising temperatures is a promising approach to generate new varieties better suited to face the environmental constraints caused by climate change. In this study, we identified several Brassica napus root traits altered in response to warm ambient temperatures. Different combinations of changes in specific root traits result in an extended and deeper root system. This overall root growth expansion facilitates root response by maximizing root-soil surface interaction and increasing roots' ability to explore extended soil areas. We associated these traits with coordinated cellular events, including changes in cell division and elongation rates that drive root growth increases triggered by warm temperatures. Comparative transcriptomic analysis revealed the main genetic determinants of these root system architecture (RSA) changes and uncovered the necessity of a tight regulation of the heat-shock stress response to adjusting root growth to warm temperatures. Our work provides a phenotypic, cellular, and genetic framework of root response to warming temperatures that will help to harness root response mechanisms for crop yield improvement under the future climatic scenario.

Plastid gene expression involves many post-transcriptional maturation steps resulting in a complex transcriptome composed of multiple isoforms. Although short-read RNA-Seq has considerably improved our understanding of the molecular mechanisms controlling these processes, it is unable to sequence full-length transcripts. This information is crucial, however, when it comes to understanding the interplay between the various steps of plastid gene expression. Here, we describe a protocol to study the plastid transcriptome using nanopore sequencing. In the leaf of Arabidopsis thaliana, with about 1.5 million strand-specific reads mapped to the chloroplast genome, we could recapitulate most of the complexity of the plastid transcriptome (polygenic transcripts, multiple isoforms associated with post-transcriptional processing) using virtual Northern blots. Even if the transcripts longer than about 2500 nucleotides were missing, the study of the co-occurrence of editing and splicing events identified 42 pairs of events that were not occurring independently. This study also highlighted a preferential chronology of maturation events with splicing happening after most sites were edited.

In many plant species, gene dosage is an important cause of phenotype variation. Engineering gene dosage, particularly in polyploid genomes, would provide an efficient tool for plant breeding. The hexaploid oilseed crop Camelina sativa, which has three closely related expressed subgenomes, is an ideal species for investigation of the possibility of creating a large collection of combinatorial mutants. Selective, targeted mutagenesis of the three delta-12-desaturase (FAD2) genes was achieved by CRISPR-Cas9 gene editing, leading to reduced levels of polyunsaturated fatty acids and increased accumulation of oleic acid in the oil. Analysis of mutations over four generations demonstrated the presence of a large variety of heritable mutations in the three isologous CsFAD2 genes. The different combinations of single, double and triple mutants in the T3 generation were isolated, and the complete loss-of-function mutants revealed the importance of delta-12-desaturation for Camelina development. Combinatorial association of different alleles for the three FAD2 loci provided a large diversity of Camelina lines with various lipid profiles, ranging from 10% to 62% oleic acid accumulation in the oil. The different allelic combinations allowed an unbiased analysis of gene dosage and function in this hexaploid species, but also provided a unique source of genetic variability for plant breeding.

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.

Understanding the mechanisms triggering variation of cell wall degradability is a prerequisite to improving the energy value of lignocellulosic biomass for animal feed or biorefinery. Here, we implemented a multiscale systems approach to shed light on the genetic basis of cell wall degradability in maize. We demonstrated that allele replacement in two pairs of near-isogenic lines at a region encompassing a major quantitative trait locus (QTL) for cell wall degradability led to phenotypic variation of a similar magnitude and sign to that expected from a QTL analysis of cell wall degradability in the F271 × F288 recombinant inbred line progeny. Using DNA sequences within the QTL interval of both F271 and F288 inbred lines and Illumina RNA sequencing datasets from internodes of the selected near-isogenic lines, we annotated the genes present in the QTL interval and provided evidence that allelic variation at the introgressed QTL region gives rise to coordinated changes in gene expression. The identification of a gene co-expression network associated with cell wall-related trait variation revealed that the favorable F288 alleles exploit biological processes related to oxidation-reduction, regulation of hydrogen peroxide metabolism, protein folding and hormone responses. Nested in modules of co-expressed genes, potential new cell-wall regulators were identified, including two transcription factors of the group VII ethylene response factor family, that could be exploited to fine-tune cell wall degradability. Overall, these findings provide new insights into the regulatory mechanisms by which a major locus influences cell wall degradability, paving the way for its map-based cloning in maize.

Mitochondria and chloroplasts are important actors in the plant nutritional efficiency. So, it could be expected that a disruption of the coadaptation between nuclear and organellar genomes impact plant response to nutrient stresses. We addressed this issue using two Arabidopsis accessions, namely Ct1 and Jea, and their reciprocal cytolines possessing the nuclear genome from one parent and the organellar genomes of the other one. We measured gene expression, and quantified proteins and metabolites under N starvation and non-limiting conditions. We observed a typical response to N starvation at the phenotype and molecular levels. The phenotypical response to N starvation was similar in the cytolines compared to the parents. However, we observed an effect of the disruption of genomic coadaptation at the molecular levels, distinct from the previously described responses to organellar stresses. Strikingly, genes differentially expressed in cytolines compared to parents were mainly repressed in the cytolines. These genes encoded more mitochondrial and nuclear proteins than randomly expected, while N starvation responsive ones were enriched in genes for chloroplast and nuclear proteins. In cytolines, the non-coadapted cytonuclear genomic combination tends to modulate the response to N starvation observed in the parental lines on various biological processes.

The vascular system of plants consists of two main tissue types, xylem and phloem. These tissues are organized into vascular bundles that are arranged into a complex network running through the plant that is essential for the viability of land plants. Despite their obvious importance, the genes involved in the organization of vascular tissues remain poorly understood in grasses.

Mycoheterotrophic plants have lost the ability to photosynthesize and obtain essential mineral and organic nutrients from associated soil fungi. Despite involving radical changes in life history traits and ecological requirements, the transition from autotrophy to mycoheterotrophy has occurred independently in many major lineages of land plants, most frequently in Orchidaceae. Yet the molecular mechanisms underlying this shift are still poorly understood. A comparison of the transcriptomes of Epipogium aphyllum and Neottia nidus-avis, two completely mycoheterotrophic orchids, to other autotrophic and mycoheterotrophic orchids showed the unexpected retention of several genes associated with photosynthetic activities. In addition to these selected retentions, the analysis of their expression profiles showed that many orthologs had inverted underground/aboveground expression ratios compared to autotrophic species. Fatty acid and amino acid biosynthesis as well as primary cell wall metabolism were among the pathways most impacted by this expression reprogramming. Our study suggests that the shift in nutritional mode from autotrophy to mycoheterotrophy remodeled the architecture of the plant metabolism but was associated primarily with function losses rather than metabolic innovations.

Very long chain fatty acids (VLCFAs) are involved in plant development and particularly in several cellular processes such as membrane trafficking, cell division and cell differentiation. However, the precise role of VLCFAs in these different cellular processes is still poorly understood in plants. In order to identify new factors associated with the biosynthesis or function of VLCFAs, a yeast multicopy suppressor screen was carried out in a yeast mutant strain defective for fatty acid elongation. Loss of function of the elongase 3 hydroxyacyl-CoA dehydratase PHS1 in yeast and PASTICCINO2 in plants prevents growth and induces cytokinesis defects. PROTEIN TYROSIN PHOSPHATASE-LIKE (PTPLA) previously characterized as an inactive dehydratase was able to restore yeast phs1 growth and VLCFAs elongation but not the plant pas2-1 defects. PTPLA interacted with elongase subunits in the Endoplasmic Reticulum (ER) and its absence induced the accumulation of 3-hydroxyacyl-CoA as expected from a dehydratase involved in fatty acid (FA) elongation. However, loss of PTPLA function increased VLCFA levels, an effect that was dependent on the presence of PAS2 indicating that PTPLA activity repressed FA elongation. The two dehydratases have specific expression profiles in the root with PAS2, mostly restricted to the endodermis, while PTPLA was confined in the vascular tissue and pericycle cells. Comparative ectopic expression of PTPLA and PAS2 in their respective domains confirmed the existence of two independent elongase complexes based on PAS2 or PTPLA dehydratase that are functionally interacting.

In higher plants, cellulose is synthesized by membrane-spanning large protein complexes named cellulose synthase complexes (CSCs). In this study, the Arabidopsis PASTICCINO2 (PAS2) was identified as an interacting partner of cellulose synthases. PAS2 was previously characterized as the plant 3-hydroxy-acyl-CoA dehydratase, an ER membrane-localized dehydratase that is essential for very-long-chain-fatty acid (VLCFA) elongation. The pas2-1 mutants show defective cell elongation and reduction in cellulose content in both etiolated hypocotyls and light-grown roots. Although disruption of VLCFA synthesis by a genetic alteration had a reduction in VLCFA in both etiolated hypocotyls and light-grown roots, it had a differential effect on cellulose content in the two systems, suggesting the threshold level of VLCFA for efficient cellulose synthesis may be different in the two biological systems. pas2-1 had a reduction in both CSC delivery rate and CSC velocity at the PM in etiolated hypocotyls. Interestingly, Golgi but not post-Golgi endomembrane structures exhibited a severe defect in motility. Experiments using pharmacological perturbation of VLCFA content in etiolated hypocotyls strongly indicate a novel function of PAS2 in the regulation of CSC and Golgi motility. Through a combination of genetic, biochemical and cell biology studies, our study demonstrated that PAS2 as a multifunction protein has an important role in the regulation of cellulose biosynthesis in Arabidopsis hypocotyl.

We address here organellar genetic regulation and intercompartment genome coordination. We developed earlier a strategy relying on a tRNA-like shuttle to mediate import of nuclear transgene-encoded custom RNAs into mitochondria in plants. In the present work, we used this strategy to drive trans-cleaving hammerhead ribozymes into the organelles, to knock down specific mitochondrial RNAs and analyze the regulatory impact. In a similar approach, the tRNA mimic was used to import into mitochondria in Arabidopsis thaliana the orf77, an RNA associated with cytoplasmic male sterility in maize and possessing sequence identities with the atp9 mitochondrial RNA. In both cases, inducible expression of the transgenes allowed to characterise early regulation and signaling responses triggered by these respective manipulations of the organellar transcriptome. The results imply that the mitochondrial transcriptome is tightly controlled by a "buffering" mechanism at the early and intermediate stages of plant development, a control that is released at later stages. On the other hand, high throughput analyses showed that knocking down a specific mitochondrial mRNA triggered a retrograde signaling and an anterograde nuclear transcriptome response involving a series of transcription factor genes and small RNAs. Our results strongly support transcriptome coordination mechanisms within the organelles and between the organelles and the nucleus.

Chromatin remodeling plays a key role in the establishment and maintenance of gene expression patterns essential for plant development and responses to environmental factors. Post-translational modification of histones, including acetylation, is one of the most relevant chromatin remodeling mechanisms that operate in eukaryotic cells. Histone acetylation is an evolutionarily conserved chromatin signature commonly associated with transcriptional activation. Histone acetylation levels are tightly regulated through the antagonistic activity of histone acetyltransferases (HATs) and histone deacetylases (HDACs). In plants, different families of HATs are present, including the MYST family, which comprises homologs of the catalytic subunit of the Nucleosome Acetyltransferase of H4 (NuA4) complex in yeast. This complex mediates acetylation of histones H4, H2A, and H2A.Z, and is involved in transcriptional regulation, heterochromatin silencing, cell cycle progression, and DNA repair in yeast. In Arabidopsis and, other plant species, homologs for most of the yeast NuA4 subunits are present and although the existence of this complex has not been demonstrated yet, compelling evidence supports the notion that this type of HAT complex functions from mosses to angiosperms. Recent proteomic studies show that several Arabidopsis homologs of NuA4 components, including the assembly platform proteins and the catalytic subunit, are associated in vivo with additional members of this complex suggesting that a NuA4-like HAT complex is present in plants. Furthermore, the functional characterization of some Arabidopsis NuA4 subunits has uncovered the involvement of these proteins in the regulation of different plant biological processes. Interestingly, for most of the mutant plants deficient in subunits of this complex characterized so far, conspicuous defects in flowering time are observed, suggesting a role for NuA4 in the control of this plant developmental program. Moreover, the participation of Arabidopsis NuA4 homologs in other developmental processes, such as gametophyte development, as well as in cell proliferation and stress and hormone responses, has also been reported. In this review, we summarize the current state of knowledge on plant putative NuA4 subunits and discuss the latest progress concerning the function of this chromatin modifying complex.

Plant defense responses involve several biological processes that allow plants to fight against pathogenic attacks. How these different processes are orchestrated within organs and depend on specific cell types is poorly known. Here, using single-cell RNA sequencing (scRNA-seq) technology on three independent biological replicates, we identified several cell populations representing the core transcriptional responses of wild-type Arabidopsis leaves inoculated with the bacterial pathogen Pseudomonas syringae DC3000. Among these populations, we retrieved major cell types of the leaves (mesophyll, guard, epidermal, companion, and vascular S cells) with which we could associate characteristic transcriptional reprogramming and regulators, thereby specifying different cell-type responses to the pathogen. Further analyses of transcriptional dynamics, on the basis of inference of cell trajectories, indicated that the different cell types, in addition to their characteristic defense responses, can also share similar modules of gene reprogramming, uncovering a ubiquitous antagonism between immune and susceptible processes. Moreover, it appears that the defense responses of vascular S cells, epidermal cells, and mesophyll cells can evolve along two separate paths, one converging toward an identical cell fate, characterized mostly by lignification and detoxification functions. As this divergence does not correspond to the differentiation between immune and susceptible cells, we speculate that this might reflect the discrimination between cell-autonomous and non-cell-autonomous responses. Altogether our data provide an upgraded framework to describe, explore, and explain the specialization and the coordination of plant cell responses upon pathogenic challenge.