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SUMMARYRNA-DNA hybrid (R-loop)-associated long noncoding RNAs (lncRNAs), including the Arabidopsis lncRNAAUXIN-REGULATED PROMOTER LOOP(APOLO), are emerging as important regulators of three-dimensional chromatin conformation and gene transcriptional activity. Here, we showed that in addition to the PRC1-component LIKE-HETEROCHROMATIN PROTEIN 1 (LHP1),APOLOinteracts with the methylcytosine-binding protein VARIANT IN METHYLATION 1 (VIM1), a conserved homolog of the mammalian DNA methylation regulator UBIQUITIN-LIKE CONTAINING PHD AND RING FINGER DOMAINS 1 (UHRF1). TheAPOLO-VIM1-LHP1 complex directly regulates the transcription of the auxin biosynthesis geneYUCCA2by dynamically determining DNA methylation and H3K27me3 deposition over its promoter during the plant thermomorphogenic response. Strikingly, we demonstrated that the lncRNAUHRF1 Protein Associated Transcript(UPAT), a direct interactor of UHRF1 in humans, can be recognized by VIM1 and LHP1 in plant cells, despite the lack of sequence homology betweenUPATandAPOLO. In addition, we showed that increased levels ofAPOLOorUPAThamper VIM1 and LHP1 binding toYUCCA2promoter. Collectively, our results uncover a new mechanism in which a plant lncRNA coordinates Polycomb action and DNA methylation, and reveal that evolutionary unrelated lncRNAs may exert similar functions across kingdoms.

Background RNA-DNA hybrid (R-loop)-associated long noncoding RNAs (lncRNAs), including the Arabidopsis lncRNA AUXIN-REGULATED PROMOTER LOOP (APOLO), are emerging as important regulators of three-dimensional chromatin conformation and gene transcriptional activity. Results Here, we show that in addition to the PRC1-component LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), APOLO interacts with the methylcytosine-binding protein VARIANT IN METHYLATION 1 (VIM1), a conserved homolog of the mammalian DNA methylation regulator UBIQUITIN-LIKE CONTAINING PHD AND RING FINGER DOMAINS 1 (UHRF1). The APOLO-VIM1-LHP1 complex directly regulates the transcription of the auxin biosynthesis gene YUCCA2 by dynamically determining DNA methylation and H3K27me3 deposition over its promoter during the plant thermomorphogenic response. Strikingly, we demonstrate that the lncRNA UHRF1 Protein Associated Transcript (UPAT), a direct interactor of UHRF1 in humans, can be recognized by VIM1 and LHP1 in plant cells, despite the lack of sequence homology between UPAT and APOLO. In addition, we show that increased levels of APOLO or UPAT hamper VIM1 and LHP1 binding to YUCCA2 promoter and globally alter the Arabidopsis transcriptome in a similar manner. Conclusions Collectively, our results uncover a new mechanism in which a plant lncRNA coordinates Polycomb action and DNA methylation through the interaction with VIM1, and indicates that evolutionary unrelated lncRNAs with potentially conserved structures may exert similar functions by interacting with homolog partners.

Plant long noncoding RNAs (lncRNAs) are key chromatin dynamics regulators, directing the transcriptional programs driving a wide variety of developmental outputs. Recently, we uncovered how the lncRNA AUXIN REGULATED PROMOTER LOOP (APOLO) directly recognizes the locus encoding the root hair (RH) master regulator ROOT HAIR DEFECTIVE 6 (RHD6) modulating its transcriptional activation and leading to low temperature-induced RH elongation. We further demonstrated that APOLO interacts with the transcription factor WRKY42 in a novel ribonucleoprotein complex shaping RHD6 epigenetic environment and integrating signals governing RH growth and development. In this work, we expand this model showing that APOLO is able to bind and positively control the expression of several cell wall EXTENSIN (EXT) encoding genes, including EXT3, a key regulator for RH growth. Interestingly, EXT3 emerged as a novel common target of APOLO and WRKY42. Furthermore, we showed that the ROS homeostasis-related gene NADPH OXIDASE C (NOXC) is deregulated upon APOLO overexpression, likely through the RHD6-RSL4 pathway, and that NOXC is required for low temperature-dependent enhancement of RH growth. Collectively, our results uncover an intricate regulatory network involving the APOLO/WRKY42 hub in the control of master and effector genes during RH development.

Plant long noncoding RNAs (lncRNAs) are key chromatin dynamics regulators, directing the transcriptional programs driving a wide variety of developmental outputs. Recently, we uncovered how the lncRNA AUXIN REGULATED PROMOTER LOOP (APOLO) directly recognizes the locus encoding the root hair (RH) master regulator ROOT HAIR DEFECTIVE 6 (RHD6) modulating its transcriptional activation and leading to low temperature-induced RH elongation. We further demonstrated that APOLO interacts with the transcription factor WRKY42 in a novel ribonucleoprotein complex shaping RHD6 epigenetic environment and integrating signals governing RH growth and development. In this work, we expand this model showing that APOLO is able to bind and positively control the expression of several cell wall EXTENSIN (EXT) encoding genes, including EXT3, a key regulator for RH growth. Interestingly, EXT3 emerged as a novel common target of APOLO and WRKY42. Furthermore, we showed that the ROS homeostasis-related gene NADPH OXIDASE C (NOXC) is deregulated upon APOLO overexpression, likely through the RHD6-RSL4 pathway, and that NOXC is required for low temperature-dependent enhancement of RH growth. Collectively, our results uncover an intricate regulatory network involving the APOLO/WRKY42 hub in the control of master and effector genes during RH development.

International audience; Plant long noncoding RNAs (lncRNAs) have emerged as important regulators of chromatin dynamics, impacting on transcriptional programs leading to different developmental outputs. The lncRNA AUXIN-REGULATED PROMOTER LOOP (APOLO) directly recognizes multiple independent loci across the Arabidopsis genome and modulates their three-dimensional chromatin conformation, leading to transcriptional shifts. Here, we show that APOLO recognizes the locus encoding the root hair (RH) master regulator ROOT HAIR DEFECTIVE 6 (RHD6) and controls RHD6 transcriptional activity, leading to cold-enhanced RH elongation through the consequent activation of the transcription factor gene RHD6-like RSL4. Furthermore, we demonstrate that APOLO interacts with the transcription factor WRKY42 and modulates its binding to the RHD6 promoter. WRKY42 is required for the activation of RHD6 by low temperatures and WRKY42 deregulation impairs cold-induced RH expansion. Collectively, our results indicate that a novel ribonucleoprotein complex with APOLO and WRKY42 forms a regulatory hub to activate RHD6 by shaping its epigenetic environment and integrate signals governing RH growth and development.

Plant long noncoding RNAs (lncRNAs) have emerged as important regulators of chromatin dynamics, impacting on transcriptional programs leading to different developmental outputs. The lncRNAAUXIN REGULATED PROMOTER LOOP(APOLO) directly recognizes multiple independent loci across theArabidopsisgenome and modulates their three-dimensional chromatin conformation, leading to transcriptional shifts. Here, we show thatAPOLOrecognizes the locus encoding the root hair (RH) master regulator ROOT HAIR DEFECTIVE 6 (RHD6) and controlsRHD6transcriptional activity leading to cold-enhanced RH elongation through the consequent activation of the transcription factor gene RHD6-likeRSL4. Furthermore, we demonstrate thatAPOLOinteracts with the transcription factor WRKY42 and modulates its binding to theRHD6promoter. WRKY42 is required for the activation ofRHD6by low temperatures andWRKY42deregulation impairs cold-induced RH expansion. Collectively, our results indicate that a novel ribonucleoprotein complex involvingAPOLOand WRKY42 forms a regulatory hub which activatesRHD6by shaping its epigenetic environment and integrates signals governing RH growth and development.SUMMARYThe lncRNAAPOLOdirectly regulates the transcription of the root hair-master geneRHD6. In response to cold,APOLOis induced and it decoys the H3K27me3-binding protein LHP1 away fromRHD6. In addition,APOLOmodulates the binding of the transcription factor WRKY42 to theRHD6promoter at low temperatures.

Alternative splicing (AS) is a major source of transcriptome diversity. Long noncoding RNAs (lncRNAs) have emerged as regulators of AS through different molecular mechanisms. In Arabidopsis thaliana, the AS regulators NSRs interact with the ALTERNATIVE SPLICING COMPETITOR (ASCO) lncRNA. Here, we analyze the effect of the knock-down and overexpression of ASCO at the genome-wide level and find a large number of deregulated and differentially spliced genes related to flagellin responses and biotic stress. In agreement, ASCO-silenced plants are more sensitive to flagellin. However, only a minor subset of deregulated genes overlaps with the AS defects of the nsra/b double mutant, suggesting an alternative way of action for ASCO. Using biotin-labeled oligonucleotides for RNA-mediated ribonucleoprotein purification, we show that ASCO binds to the highly conserved spliceosome component PRP8a. ASCO overaccumulation impairs the recognition of specific flagellin-related transcripts by PRP8a. We further show that ASCO also binds to another spliceosome component, SmD1b, indicating that it interacts with multiple splicing factors. Hence, lncRNAs may integrate a dynamic network including spliceosome core proteins, to modulate transcriptome reprogramming in eukaryotes. Fil: Rigo, Richard. Centre National de la Recherche Scientifique; Francia Fil: Bazin, Jérémie. Centre National de la Recherche Scientifique; Francia Fil: Romero Barrios, Natali. Centre National de la Recherche Scientifique; Francia Fil: Moison, Michael. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Lucero, Leandro Exequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Christ, Aurelie. Centre National de la Recherche Scientifique; Francia Fil: Benhamed, Moussa. Centre National de la Recherche Scientifique; Francia Fil: Blein, Thomas. Centre National de la Recherche Scientifique; Francia Fil: Huguet, Stephanie. Centre National de la Recherche Scientifique; Francia Fil: Charon, Celine. Centre National de la Recherche Scientifique; Francia Fil: Crespi, Martin. Centre National de la Recherche Scientifique; Francia Fil: Ariel, Federico Damian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina

Glutamine biosynthesis for N-remobilization and seed filling in Arabidopsis is mainly catalysed by the three major GS1 isoforms, GLN1;1, GLN1;2, and GLN1;3, which are localized in different-order veins in the leaves., Glutamine synthetase (GS) is central for ammonium assimilation and consists of cytosolic (GS1) and chloroplastic (GS2) isoenzymes. During plant ageing, GS2 protein decreases due to chloroplast degradation, and GS1 activity increases to support glutamine biosynthesis and N remobilization from senescing leaves. The role of the different Arabidopsis GS1 isoforms in nitrogen remobilization was examined using 15N tracing experiments. Only the gln1;1-gln1;2-gln1;3 triple-mutation affecting the three GLN1;1, GLN1;2, and GLN1;3 genes significantly reduced N remobilization, total seed yield, individual seed weight, harvest index, and vegetative biomass. The triple-mutant accumulated a large amount of ammonium that could not be assimilated by GS1. Alternative ammonium assimilation through asparagine biosynthesis was increased and was related to higher ASN2 asparagine synthetase transcript levels. The GS2 transcript, protein, and activity levels were also increased to compensate for the lack of GS1-related glutamine biosynthesis. Localization of the different GLN1 genes showed that they were all expressed in the phloem companion cells but in veins of different order. Our results demonstrate that glutamine biosynthesis for N-remobilization occurs in veins of all orders (major and minor) in leaves, it is mainly catalysed by the three major GS1 isoforms (GLN1;1, GLN1;2, and GLN1;3), and it is alternatively supported by AS2 in the veins and GS2 in the mesophyll cells.

Metabolomics of leaf laminae and veins during ageing reveal tissue specificities and different senescence programmes., Leaf senescence is a long developmental process important for nutrient management and for source to sink remobilization. Constituents of the mesophyll cells are progressively degraded to provide nutrients to the rest of the plant. Up to now, studies on leaf senescence have not paid much attention to the role of the different leaf tissues. In the present study, we dissected leaf laminae from the midvein to perform metabolite profiling. The laminae mesophyll cells are the source of nutrients, and in C3 plants they contain Rubisco as the most important nitrogen storage pool. Veins, rich in vasculature, are the place where all the nutrients are translocated, and sometimes interconverted, before being exported through the phloem or the xylem. The different metabolic changes we observed in laminae and midvein with ageing support the idea that the senescence programme in these two tissues is different. Important accumulations of metabolites in the midvein suggest that nutrient translocations from source leaves to sinks are mainly controlled at this level. Carbon and nitrogen long-distance molecules such as fructose, glucose, aspartate, and asparagine were more abundant in the midvein than in laminae. In contrast, sucrose, glutamate, and aspartate were more abundant in laminae. The concentrations of tricarboxylic acid (TCA) compounds were also lower in the midvein than in laminae. Since nitrogen remobilization increased under low nitrate supply, plants were grown under two nitrate concentrations. The results revealed that the senescence-related differences were mostly similar under low and high nitrate conditions except for some pathways such as the TCA cycle.

Large numbers of publications have appeared over the last few years, dealing with the molecular details of the regulation and process of the autophagy machinery in animals, plants, and unicellular eukaryotic organisms. This strong interest is caused by the fact that the autophagic process is involved in the adaptation of organisms to their environment and to stressful conditions, thereby contributing to cell and organism survival and longevity. In plants, as in other eukaryotes, autophagy is associated with longevity as mutants display early and strong leaf senescence symptoms, however, the exact role of autophagy as a pro-survival or pro-death process is unclear. Recently, evidence that autophagy participates in nitrogen remobilization has been provided, but the duality of the role of autophagy in leaf longevity and/or nutrient recycling through cell component catabolism remains. This review aims to give an overview of leaf senescence-associated processes from the physiological point of view and to discuss relationships between nutrient recycling, proteolysis, and autophagy. The dual role of autophagy as a pro-survival or pro-death process is discussed.

Summary Legume crops related to the model plant Medicago truncatula can adapt their root architecture to environmental conditions, both by branching and by establishing a symbiosis with rhizobial bacteria to form nitrogen-fixing nodules. Soil salinity is a major abiotic stress affecting plant yield and root growth. Previous transcriptomic analyses identified several transcription factors linked to the M. truncatula response to salt stress in roots, including NAC (NAM/ATAF/CUC)-encoding genes. Over-expression of one of these transcription factors, MtNAC969, induced formation of a shorter and less-branched root system, whereas RNAi-mediated MtNAC969 inactivation promoted lateral root formation. The altered root system of over-expressing plants was able to maintain its growth under high salinity, and roots in which MtNAC969 was down-regulated showed improved growth under salt stress. Accordingly, expression of salt stress markers was decreased or induced in MtNAC969 over-expressing or RNAi roots, respectively, suggesting a repressive function for this transcription factor in the salt-stress response. Expression of MtNAC969 in central symbiotic nodule tissues was induced by nitrate treatment, and antagonistically affected by salt in roots and nodules, similarly to senescence markers. MtNAC969 RNAi nodules accumulated amyloplasts in the nitrogen-fixing zone, and were prematurely senescent. Therefore, the MtNAC969 transcription factor, which is differentially affected by environmental cues in root and nodules, participates in several pathways controlling adaptation of the M. truncatula root system to the environment.

In recent years Arabidopsis thaliana has become a model species for genomic variability and adaptation studies. Although impressive quantities of data have been gathered on the nuclear genomic diversity of this species, little has been published regarding its cytoplasmic diversity. We analyzed the diversity of plastid (pt) and mitochondrial (mt) genomes among 95 accessions, covering most Arabidopsis geographic origins. Four intergenic regions of the pt genome were sequenced, and a total of 68 polymorphisms and 65 pt haplotypes were identified. Several strategies were developed to identify mt polymorphisms among a subset of 14 accessions. Fifteen polymorphisms were further developed as PCR-based markers and used to analyze the whole set of 95 accessions. Using statistical parsimony, we built pt and mt phylogenetic networks of haplotype groups. To root the pt network, the pt intergenic regions of two related Arabidopsis species, Arabidopsis lyrata and Arabidopsis arenosa, were also sequenced. The mt and pt phylogenies are highly congruent and could be combined into a single cytoplasmic phylogeny. To estimate whether co-adaptation between nuclear and cytoplasmic genomes exists in A. thaliana, we tested the germination capacity in challenging conditions of 27 pairs of reciprocal F(2) families. We found that the cytoplasm donor had a significant effect on the germination capacity of some F(2) families.

In the absence of cell migration, the orientation of cell divisions is crucial for body plan determination in plants. The position of the division plane in plant cells is set up premitotically via a transient cytoskeletal array, the preprophase band, which precisely delineates the cortical plane of division. Here we describe a protein complex that targets protein phosphatase 2A activity to microtubules, regulating the transition from the interphase to the premitotic microtubule array. This complex, which comprises TONNEAU1 and a PP2A heterotrimeric holoenzyme with FASS as regulatory subunit, is recruited to the cytoskeleton via the TONNEAU1-recruiting motif family of proteins. Despite the acentrosomal nature of plant cells, all members of this complex share similarity with animal centrosomal proteins involved in ciliary and centriolar/centrosomal functions, revealing an evolutionary link between the cortical cytoskeleton of plant cells and microtubule organizers in other eukaryotes.

Cytokinin regulates many aspects of plant development, and in legume crops, this phytohormone is necessary and sufficient for symbiotic nodule organogenesis, allowing them to fix atmospheric nitrogen. To identify direct links between cytokinins and nodule organogenesis, we determined a consensus sequence bound in vitro by a transcription factor (TF) acting in cytokinin signaling, the nodule-enhanced Medicago truncatula Mt RR1 response regulator (RR). Among genes rapidly regulated by cytokinins and containing this so-called RR binding site (RRBS) in their promoters, we found the nodulation-related Type-A RR Mt RR4 and the Nodulation Signaling Pathway 2 (NSP2) TF. Site-directed mutagenesis revealed that RRBS cis-elements in the RR4 and NSP2 promoters are essential for expression during nodule development and for cytokinin induction. Furthermore, a microRNA targeting NSP2 (miR171 h) is also rapidly induced by cytokinins and then shows an expression pattern anticorrelated with NSP2. Other primary targets regulated by cytokinins depending on the Cytokinin Response1 (CRE1) receptor were a cytokinin oxidase/dehydrogenase (CKX1) and a basic Helix-Loop-Helix TF (bHLH476). RNA interference constructs as well as insertion of a Tnt1 retrotransposon in the bHLH gene led to reduced nodulation. Hence, we identified two TFs, NSP2 and bHLH476, as direct cytokinin targets acting at the convergence of phytohormonal and symbiotic cues.

In recent years Arabidopsis thaliana has become a model species for genomic variability and adaptation studies. Although impressive quantities of data have been gathered on the nuclear genomic diversity of this species, little has been published regarding its cytoplasmic diversity. We analyzed the diversity of plastid (pt) and mitochondrial (mt) genomes among 95 accessions, covering most Arabidopsis geographic origins. Four intergenic regions of the pt genome were sequenced, and a total of 68 polymorphisms and 65 pt haplotypes were identified. Several strategies were developed to identify mt polymorphisms among a subset of 14 accessions. Fifteen polymorphisms were further developed as PCR-based markers and used to analyze the whole set of 95 accessions. Using statistical parsimony, we built pt and mt phylogenetic networks of haplotype groups. To root the pt network, the pt intergenic regions of two related Arabidopsis species, Arabidopsis lyrata and Arabidopsis arenosa, were also sequenced. The mt and pt phylogenies are highly congruent and could be combined into a single cytoplasmic phylogeny. To estimate whether co-adaptation between nuclear and cytoplasmic genomes exists in A. thaliana, we tested the germination capacity in challenging conditions of 27 pairs of reciprocal F(2) families. We found that the cytoplasm donor had a significant effect on the germination capacity of some F(2) families.