Lorena A. Siena

Characterization and expression analysis of SOMATIC EMBRYOGENESIS RECEPTOR KINASE ( SERK ) genes in sexual and apomictic Paspalum notatum

The SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) gene plays a fundamental role in somatic embryogenesis of angiosperms, and is associated with apomixis in Poa pratensis. The objective of this work was to isolate, characterize and analyze the expression patterns of SERK genes in apomictic and sexual genotypes of Paspalum notatum. A conserved 200-bp gene fragment was amplified from genomic DNA with heterologous primers, and used to initiate a chromosomal walking strategy for cloning the complete sequence. This procedure allowed the isolation of two members of the P. notatumSERK family; PnSERK1, which is similar to PpSERK1, and PnSERK2, which is similar to ZmSERK2 and AtSERK1. Phylogenetic analyses indicated that PnSERK1 and PnSERK2 represent paralogous sequences. Southern-blot hybridization indicated the presence of at least three copies of SERK genes in the species. qRT-PCR analyses revealed that PnSERK2 was expressed at significantly higher levels than PnSERK1 in roots, leaves, reproductive tissues and embryogenic calli. Moreover, in situ hybridization experiments revealed that PnSERK2 displayed a spatially and chronologically altered expression pattern in reproductive organs of the apomictic genotype with respect to the sexual one. PnSERK2 is expressed in nucellar cells of the apomictic genotype at meiosis, but only in the megaspore mother cell in the sexual genotype. Therefore, apomixis onset in P. notatum seems to be correlated with the expression of PnSERK2 in nucellar tissue.

Sequence characterization, in silico mapping and cytosine methylation analysis of markers linked to apospory in Paspalum notatum

In previous studies we reported the identification of several AFLP, RAPD and RFLP molecular markers linked to apospory in Paspalum notatum. The objective of this work was to sequence these markers, obtain their flanking regions by chromosome walking and perform an in silico mapping analysis in rice and maize. The methylation status of two apospory-related sequences was also assessed using methylation-sensitive RFLP experiments. Fourteen molecular markers were analyzed and several protein-coding sequences were identified. Copy number estimates and RFLP linkage analysis showed that the sequence PnMAI3 displayed 2–4 copies per genome and linkage to apospory. Extension of this marker by chromosome walking revealed an additional protein-coding sequence mapping in silico in the apospory-syntenic regions of rice and maize. Approximately 5 kb corresponding to different markers were characterized through the global sequencing procedure. A more refined analysis based on sequence information indicated synteny with segments of chromosomes 2 and 12 of rice and chromosomes 3 and 5 of maize. Two loci associated with apomixis locus were tested in methylation-sensitive RFLP experiments using genomic DNA extracted from leaves. Although both target sequences were methylated no methylation polymorphisms associated with the mode of reproduction were detected.

An apomixis-linked ORC3-like pseudogene is associated with silencing of its functional homolog in apomictic Paspalum simplex

Apomixis in plants consists of asexual reproduction by seeds. Here we characterized at structural and functional levels an apomixis-linked sequence of Paspalum simplex homologous to subunit 3 of the ORIGIN RECOGNITION COMPLEX (ORC3). ORC is a multiprotein complex which controls DNA replication and cell differentiation in eukaryotes. Three PsORC3 copies were identified, each one characterized by a specific expression profile. Of these, PsORC3a, specific for apomictic genotypes, is a pseudogene that was poorly and constitutively expressed in all developmental stages of apomictic flowers, whereas PsORC3b, the putative functional gene in sexual flowers, showed a precise time-related regulation. Sense transcripts of PsORC3 were expressed in the female cell lineage of both apomictic and sexual reproductive phenotypes, and in aposporous initials. Although strong expression was detected in sexual early endosperm, no expression was present in the apomictic endosperm. Antisense PsORC3 transcripts were revealed exclusively in apomictic germ cell lineages. Defective orc3 mutants of rice and Arabidopsis showed normal female gametophytes although the embryo and endosperm were arrested at early phases of development. We hypothesize that PsORC3a is associated with the down-regulation of its functional homolog and with the development of apomictic endosperm which deviates from the canonical 2(maternal):1(paternal) genome ratio.

Expression of lorelei-like genes in aposporous and sexual Paspalum notatum plants

Gametophytic apomictic plants form non-reduced embryo sacs that generate clonal embryos by parthenogenesis, in the absence of both meiosis and egg-cell fertilization. Here we report the sequence and expression analysis of a lorelei-like Paspalum notatum gene, n20gap-1, which encodes a GPI-anchored protein previously associated with apomixis in this species. Phylogeny trees showed that n20gap-1 was evolutionary related to the Arabidopsisthaliana lorelei genes At4g26466 and At5g56170. The lorelei At4g26466 disruption was shown to be detrimental to sperm cell release in arabidopsis. RFLP (Restriction Fragment Length Polymorphism) analysis revealed the occurrence of several homologous sequences in the Paspalum notatum genome, exhibiting polymorphisms genetically linked to apomixis. Real-time PCR showed that lorelei-family genes present a minor activity peak at pre-meiosis and a major one at anthesis. The apomictic genotype analyzed showed a significantly increased activity at pre-meiosis, post-meiosis and anthesis with respect to a sexual genotype. In situ hybridization assays revealed expression in integuments, nucellus and the egg-cell apparatus. Several n20gap-1 alleles differing mainly at the 3′ UTR sequence were identified. Allele-specific real-time PCR experiments showed that allele 28 was significantly induced in reproductive tissues of the apomictic genotype with respect to the sexual genotype at anthesis. Our results indicate that P. notatumlorelei-like genes are differentially expressed in representative sexual (Q4188) and apomictic (Q4117) genotypes, and might play a role in the final stages of the apomixis developmental cascade. However, the association of n20gap-1 expression with the trait should be confirmed in significant number of sexual and apomictic genotypes.

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

BackgroundIn flowering plants, apomixis (asexual reproduction via seeds) is widely believed to result from failure of key regulators of the sexual female reproductive pathway. In the past few years, both differential display and RNA-seq comparative approaches involving reproductive organs of sexual plants and their apomictic counterparts have yielded extensive lists of candidate genes. Nevertheless, only a limited number of these genes have been functionally characterized, with few clues consequently available for understanding the molecular control of apomixis. We have previously identified several cDNA fragments with high similarity to genes involved in RNA biology and with differential amplification between sexual and apomictic Paspalum notatum plants. Here, we report the characterization of one of these candidates, namely, N69 encoding a protein of the S-adenosyl-L-methionine-dependent methyltransferases superfamily. The purpose of this work was to extend the N69 cDNA sequence and to characterize its expression at different developmental stages in both sexual and apomictic individuals.ResultsMolecular characterization of the N69 cDNA revealed homology with genes encoding proteins similar to yeast and mammalian trimethylguanosine synthase/PRIP-interacting proteins. These proteins play a dual role as ERK2-controlled transcriptional coactivators and mediators of sn(o)RNA and telomerase RNA cap trimethylation, and participate in mammals and yeast development. The N69-extended sequence was consequently renamed PnTgs1-like. Expression of PnTgs1-like during reproductive development was significantly higher in floral organs of sexual genotypes compared with apomicts. This difference was not detected in vegetative tissues. In addition, expression levels in reproductive tissues of several genotypes were negatively correlated with facultative apomixis rates. Moreover, in situ hybridization observations revealed that PnTgs1-like expression 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 of aposporous initials differentiation in apomictic genotypes.ConclusionsOur results indicate that PnTgs1-like (formerly N69) encodes a trimethylguanosine synthase-like protein whose function in mammals and yeast is critical for development, including reproduction. Our findings also suggest a pivotal role for this candidate gene in nucellar cell fate, as its diminished expression is correlated with initiation of the apomictic pathway in plants.

A reference floral transcriptome of sexual and apomictic Paspalum notatum

BackgroundPaspalum notatum Flügge is a subtropical grass native to South America, which includes sexual diploid and apomictic polyploid biotypes. In the past decade, a number of apomixis-associated genes were discovered in this species through genetic mapping and differential expression surveys. However, the scarce information on Paspalum sequences available in public databanks limited annotations and functional predictions for these candidates.ResultsWe used a long-read 454/Roche FLX+ sequencing strategy to produce robust reference transcriptome datasets from florets of sexual and apomictic Paspalum notatum genotypes and delivered a list of transcripts showing differential representation in both reproductive types. Raw data originated from floral samples collected from premeiosis to anthesis was assembled in three libraries: i) sexual (SEX), ii) apomictic (APO) and iii) global (SEX + APO). A group of physically-supported Paspalum mRNA and EST sequences matched with high level of confidence to both sexual and apomictic libraries. A preliminary trial allowed discovery of the whole set of putative alleles/paralogs corresponding to 23 previously identified apomixis-associated candidate genes. Moreover, a list of 3,732 transcripts and several co-expression and protein –protein interaction networks associated with apomixis were identified.ConclusionsThe use of the 454/Roche FLX+ transcriptome database will allow the detailed characterization of floral alleles/paralogs of apomixis candidate genes identified in prior and future work. Moreover, it was used to reveal additional candidate genes differentially represented in apomictic and sexual flowers. Gene ontology (GO) analyses of this set of transcripts indicated that the main molecular pathways altered in the apomictic genotype correspond to specific biological processes, like biotic and abiotic stress responses, growth, development, cell death and senescence. This data collection will be of interest to the plant reproduction research community and, particularly, to Paspalum breeding projects.

The MAP3K-coding QUI-GON JINN (QGJ) Gene Is Essential To The Formation of Unreduced Embryo Sacs In Paspalum

Apomixis is a clonal mode of reproduction via seeds, which results from the failure of meiosis and fertilization in the sexual female reproductive pathway. In previous transcriptomic surveys, we identified a mitogen-activated protein kinase kinase kinase (N46) displaying differential representation in florets of sexual and apomictic Paspalum notatum genotypes. Here, we retrieved and characterized the N46 full cDNA sequence from sexual and apomictic floral transcriptomes. Phylogenetic analyses showed that N46 was a member of the YODA family, which was re-named QUI-GON JINN (QGJ). Differential expression in florets of sexual and apomictic plants was confirmed by qPCR. In situ hybridization experiments revealed expression in the nucellus of aposporous plants’ ovules, which was absent in sexual plants. RNAi inhibition of QGJ expression in two apomictic genotypes resulted in significantly reduced rates of aposporous embryo sac formation, with respect to the level detected in wild type aposporous plants and transformation controls. The QGJ locus segregated independently of apospory. However, a probe derived from a related long non-coding RNA sequence (PN_LNC_QGJ) revealed RFLP bands cosegregating with the Paspalum apospory-controlling region (ACR). PN_LNC_QGJ is expressed in florets of apomictic plants only. Our results indicate that the activity of QGJ in the nucellus of apomictic plants is necessary to form non-reduced embryo sacs and that a long non-coding sequence with regulatory potential is similar to sequences located within the ACR.

Temporal and spatial expression of genes involved in DNA methylation during reproductive development of sexual and apomictic Eragrostis curvula

Recent reports in model plant species have highlighted a role for DNA methylation pathways in the regulation of the somatic-to-reproductive transition in the ovule, suggesting that apomixis (asexual reproduction through seeds) likely relies on RdDM downregulation. Our aim was therefore to explore this hypothesis by characterizing genes involved in DNA methylation in the apomictic grass Eragrostis curvula. We explored floral transcriptomes to identify homologs of three candidate genes, for which mutations in Arabidopsis and maize mimic apomixis (AtAGO9/ZmAGO104, AtCMT3/ZmDMT102/ZmDMT105, and AtDDM1/ZmCHR106), and compared both their spatial and temporal expression patterns during reproduction in sexual and apomictic genotypes. Quantitative expression analyses revealed contrasting expression patterns for the three genes in apomictic vs sexual plants. In situ hybridization corroborated these results for two candidates, EcAGO104 and EcDMT102, and revealed an unexpected ectopic pattern for the AGO gene during germ line differentiation in apomicts. Although our data partially support previous results obtained in sexual plant models, they suggest that rather than an RdDM breakdown in the ovule, altered localization of AtAGO9/ZmAGO104 expression is required for achieving diplospory in E. curvula. The differences in the RdDM machinery acquired during plant evolution might have promoted the emergence of the numerous apomictic paths observed in plants.