PnTgs1-like expression during reproductive development supports a role for RNA methyltransferases in the aposporous pathway

International audienceBackground:In flowering plants, apomixis (asexual reproduction via seeds) is widely believed to result from failureof key regulators of the sexual female reproductive pathway. In the past few years, both differential display andRNA-seq comparative approaches involving reproductive organs of sexual plants and their apomictic counterparts haveyielded extensive lists of candidate genes. Nevertheless, only a limited number of these genes have been functionallycharacterized, with few clues consequently available for understanding the molecular control of apomixis. We havepreviously identified several cDNA fragments with high similarity to genes involved in RNA biology and with differentialamplification between sexual and apomicticPaspalum notatumplants. Here, we report the characterization of one ofthese candidates, namely, N69 encoding a protein of the S-adenosyl-L-methionine-dependent methyltransferasessuperfamily. The purpose of this work was to extend the N69 cDNA sequence and to characterize its expression atdifferent developmental stages in both sexual and apomictic individuals.Results:Molecular characterization of the N69 cDNA revealed homology with genes encoding proteins similar to yeastand mammalian trimethylguanosine synthase/PRIP-interacting proteins. These proteins play a dual role as ERK2-controlledtranscriptional coactivators and mediators of sn(o)RNA and telomerase RNA cap trimethylation, and participate inmammals and yeast development. The N69-extended sequence was consequently renamedPnTgs1-like.ExpressionofPnTgs1-likeduring reproductive development was significantly higher in floral organs of sexual genotypes comparedwith apomicts. This difference was not detected in vegetative tissues. In addition, expression levels in reproductivetissues of several genotypes were negatively correlated with facultative apomixis rates. Moreover,in situhybridizationobservations revealed thatPnTgs1-likeexpression is relatively higher in ovules of sexual plants throughout development,from premeiosis to maturity. Tissues where differential expression is detected include nucellar cells, the site ofaposporous initials differentiation in apomictic genotypes.Conclusions:Our results indicate thatPnTgs1-like(formerly N69) encodes a trimethylguanosine synthase-likeprotein whose function in mammals and yeast is critical for development, including reproduction. Our findingsalso suggest a pivotal role for this candidate gene in nucellar cell fate, as its diminished expression is correlated withinitiation of the apomictic pathway in plants

Spotting the Targets of the Apospory Controller TGS1 in Paspalum notatum

International audienceSexuality and apomixis are interconnected plant reproductive routes possibly behaving as polyphenic traits under the influence of the environment. In the subtropical grass Paspalum notatum, one of the controllers of apospory, a main component of gametophytic apomixis reproduction, is TRIMETHYLGUANOSINE SYNTHASE 1 (TGS1), a multifunctional gene previously associated with RNA cleavage regulation (including mRNA splicing as well as rRNA and miRNA processing), transcriptional modulation and the establishment of heterochromatin. In particular, the downregulation of TGS1 induces a sexuality decline and the emergence of aposporous-like embryo sacs. The present work was aimed at identifying TGS1 target RNAs expressed during reproductive development of Paspalum notatum. First, we mined available RNA databases originated from spikelets of sexual and apomictic plants, which naturally display a contrasting TGS1 representation, to identify differentially expressed mRNA splice variants and miRNAs. Then, the role of TGS1 in the generation of these particular molecules was investigated in antisense tgs1 sexual lines. We found that CHLOROPHYLL A-B BINDING PROTEIN 1B-21 (LHC Ib-21, a component of the chloroplast light harvesting complex), QUI-GON JINN (QGJ, encoding a MAP3K previously associated with apomixis) and miR2275 (a meiotic 24-nt phasi-RNAs producer) are directly or indirectly targeted by TGS1. Our results point to a coordinated control exercised by signal transduction and siRNA machineries to induce the transition from sexuality to apomixis

Spotting the Targets of the Apospory Controller TGS1 in Paspalum notatum

Sexuality and apomixis are interconnected plant reproductive routes possibly behaving as polyphenic traits under the influence of the environment. In the subtropical grass Paspalum notatum, one of the controllers of apospory, a main component of gametophytic apomixis reproduction, is TRIMETHYLGUANOSINE SYNTHASE 1 (TGS1), a multifunctional gene previously associated with RNA cleavage regulation (including mRNA splicing as well as rRNA and miRNA processing), transcriptional modulation and the establishment of heterochromatin. In particular, the downregulation of TGS1 induces a sexuality decline and the emergence of aposporous-like embryo sacs. The present work was aimed at identifying TGS1 target RNAs expressed during reproductive development of Paspalum notatum. First, we mined available RNA databases originated from spikelets of sexual and apomictic plants, which naturally display a contrasting TGS1 representation, to identify differentially expressed mRNA splice variants and miRNAs. Then, the role of TGS1 in the generation of these particular molecules was investigated in antisense tgs1 sexual lines. We found that CHLOROPHYLL A-B BINDING PROTEIN 1B-21 (LHC Ib-21, a component of the chloroplast light harvesting complex), QUI-GON JINN (QGJ, encoding a MAP3K previously associated with apomixis) and miR2275 (a meiotic 24-nt phasi-RNAs producer) are directly or indirectly targeted by TGS1. Our results point to a coordinated control exercised by signal transduction and siRNA machineries to induce the transition from sexuality to apomixis

A Portion of the Apomixis Locus of Paspalum Simplex is Microsyntenic with an Unstable Chromosome Segment Highly Conserved Among Poaceae

Abstract The introgression of apomixis in major seed crops, would guarantee self-seeding of superior heterotic seeds over generations. In the grass species Paspalum simplex, apomixis is controlled by a single locus in which recombination is blocked. In the perspective of isolating the genetic determinants of apomixis, we report data on sequencing, in silico mapping and expression analysis of some of the genes contained in two cloned genomic regions of the apomixis locus of P. simplex. In silico mapping allowed us to identify a conserved synteny group homoeologous to the apomixis locus, located on a telomeric position of chromosomes 12, 8, 3 and 4 of rice, Sorghum bicolor, Setaria italica and Brachypodium distachyum, respectively, and on a more centromeric position of maize chromosome 1. Selected genes of the apomixis locus expressed sense and antisense transcripts in reproductively committed cells of sexual and apomictic ovules. Some of the genes considered here expressed apomixis-specific allelic variants which showed partial non-overlapping expression patterns with alleles shared by sexual and apomictic reproductive phenotypes. Our findings open new routes for the isolation of the genetic determinants of apomixis and, in perspective, for its introgression in crop grasses

TRIMETHYLGUANOSINE SYNTHASE1 mutations decanalize female germline development in Arabidopsis

International audienceSummary Here, we report the characterization of a plant RNA methyltransferase, orthologous to yeast trimethylguanosine synthase1 (Tgs1p) and whose downregulation was associated with apomixis in Paspalum grasses. Using phylogenetic analyses and yeast complementation, we determined that land plant genomes all encode a conserved, specific TGS1 protein. Next, we studied the role of TGS1 in female reproduction using reporter lines and loss‐of‐function mutants in Arabidopsis thaliana . pAtTGS1:AtTGS1 reporters showed a dynamic expression pattern. They were highly active in the placenta and ovule primordia at emergence but, subsequently, showed weak signals in the nucellus. Although expressed throughout gametophyte development, activity became restricted to the female gamete and was also detected after fertilization during embryogenesis. TGS1 depletion altered the specification of the precursor cells that give rise to the female gametophytic generation and to the sporophyte, resulting in the formation of a functional aposporous‐like lineage. Our results indicate that TGS1 participates in the mechanisms restricting cell fate acquisition to a single cell at critical transitions throughout the female reproductive lineage and, thus, expand our current knowledge of the mechanisms governing female reproductive fate in plants

Chromosome-scale genome assembly and annotation of Paspalum notatum Flüggé var. saurae

International audienceAbstract Paspalum notatum Flüggé is an economically important subtropical fodder grass that is widely used in the Americas. Here, we report a new chromosome-scale genome assembly and annotation of a diploid biotype collected in the center of origin of the species. Using Oxford Nanopore long reads, we generated a 557.81 Mb genome assembly (N50 = 56.1 Mb) with high gene completeness (BUSCO = 98.73%). Genome annotation identified 320 Mb (57.86%) of repetitive elements and 45,074 gene models, of which 36,079 have a high level of confidence. Further characterisation included the identification of 59 miRNA precursors together with their putative targets. The present work provides a comprehensive genomic resource for P. notatum improvement and a reference frame for functional and evolutionary research within the genus

The MAP3K-Coding QUI-GON JINN (QGJ) gene is essential to the formation of unreduced embryo sacs in Paspalum.

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