Dataset Information

Structure, target-specificity and expression of PN_LNC_N13, a long non-coding RNA differentially expressed in apomictic and sexual Paspalum notatum.

ABSTRACT: KEY MESSAGE: ncRNA PN_LNC_N13 shows contrasting expression in reproductive organs of sexual and apomictic Paspalum notatum genotypes. Apomictic plants set genetically maternal seeds whose embryos derive by parthenogenesis from unreduced egg cells, giving rise to clonal offspring. Several Paspalum notatum apomixis related genes were identified in prior work by comparative transcriptome analyses. Here, one of these candidates (namely N13) was characterized. N13 belongs to a Paspalum gene family including 30-60 members, of which at least eight are expressed at moderate levels in florets. The sequences of these genes show no functional ORFs, but include segments of different protein coding genes. Particularly, N13 shows partial identity to maize gene BT068773 (RESPONSE REGULATOR 6). Secondary structure predictions as well as mature miRNA and target cleavage detection suggested that N13 is not a miRNA precursor. Moreover, N13 family members produce abundant 24-nucleotide small RNAs along extensive parts of their sequences. Surveys in the GREENC and CANTATA databases indicated similarity with plant long non-coding RNAs (lncRNAs) involved in splicing regulation; consequently, N13 was renamed as PN_LNC_N13. The Paspalum BT068773 predicted ortholog (N13TAR) originates floral transcript variants shorter than the canonical maize isoform and with possible structural differences between the apomictic and sexual types. PN_LNC_N13 is expressed only in apomictic plants and displays quantitative representation variation across reproductive developmental stages. However, PN_LNC_N13-like homologs and/or its derived sRNAs showed overall a higher representation in ovules of sexual plants at late premeiosis. Our results suggest the existence of a whole family of N13-like lncRNAs possibly involved in splicing regulation, with some members characterized by differential activity across reproductive types.

SUBMITTER: Ochogavia A

PROVIDER: S-EPMC5778186 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Structure, target-specificity and expression of PN_LNC_N13, a long non-coding RNA differentially expressed in apomictic and sexual Paspalum notatum.

Ochogavía Ana A Galla Giulio G Seijo José Guillermo JG González Ana María AM Bellucci Michele M Pupilli Fulvio F Barcaccia Gianni G Albertini Emidio E Pessino Silvina S

Plant molecular biology 20171108 1-2

KEY MESSAGE: ncRNA PN_LNC_N13 shows contrasting expression in reproductive organs of sexual and apomictic Paspalum notatum genotypes. Apomictic plants set genetically maternal seeds whose embryos derive by parthenogenesis from unreduced egg cells, giving rise to clonal offspring. Several Paspalum notatum apomixis related genes were identified in prior work by comparative transcriptome analyses. Here, one of these candidates (namely N13) was characterized. N13 belongs to a Paspalum gene family in ...[more]

PMID: 29119346

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Project description: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.

Project description:BackgroundApomixis, an asexual mode of plant reproduction, is a genetically heritable trait evolutionarily related to sexuality, which enables the fixation of heterozygous genetic combinations through the development of maternal seeds. Recently, reference floral transcriptomes were generated from sexual and apomictic biotypes of Paspalum notatum, one of the most well-known plant models for the study of apomixis. However, the transcriptome dynamics, the occurrence of apomixis vs. sexual expression heterochronicity across consecutive developmental steps and the orientation of transcription (sense/antisense) remain unexplored.ResultsWe produced 24 Illumina TruSeq®/ Hiseq 1500 sense/antisense floral transcriptome libraries covering four developmental stages (premeiosis, meiosis, postmeiosis, and anthesis) in biological triplicates, from an obligate apomictic and a full sexual genotype. De novo assemblies with Trinity yielded 103,699 and 100,114 transcripts for the apomictic and sexual samples respectively. A global comparative analysis involving reads from all developmental stages revealed 19,352 differentially expressed sense transcripts, of which 13,205 (68%) and 6147 (32%) were up- and down-regulated in apomictic samples with respect to the sexual ones. Interestingly, 100 differentially expressed antisense transcripts were detected, 55 (55%) of them up- and 45 (45%) down-regulated in apomictic libraries. A stage-by-stage comparative analysis showed a higher number of differentially expressed candidates due to heterochronicity discrimination: the highest number of differential sense transcripts was detected at premeiosis (23,651), followed by meiosis (22,830), postmeiosis (19,100), and anthesis (17,962), while the highest number of differential antisense transcripts were detected at anthesis (495), followed by postmeiosis (164), meiosis (120) and premeiosis (115). Members of the AP2, ARF, MYB and WRKY transcription factor families, as well as the auxin, jasmonate and cytokinin plant hormone families appeared broadly deregulated. Moreover, the chronological expression profile of several well-characterized apomixis controllers was examined in detail.ConclusionsThis work provides a quantitative sense/antisense gene expression catalogue covering several subsequent reproductive developmental stages from premeiosis to anthesis for apomictic and sexual P. notatum, with potential to reveal heterochronic expression between reproductive types and discover sense/antisense mediated regulation. We detected a contrasting transcriptional and hormonal control in apomixis and sexuality as well as specific sense/antisense modulation occurring at the onset of parthenogenesis.

Project description:Aposporous apomictic plants form clonal maternal seeds by inducing the emergence of non-reduced (2n) embryo sacs in the ovule nucellus and the development of embryos by parthenogenesis. In previous work, we reported a plant-specific TRIMETHYLGUANOSINE SYNTHASE 1 (TGS1) gene (PN_TGS1-like) showing expression levels positively correlated with sexuality rates in facultative apomictic Paspalum notatum. PN_ TGS1-like displayed contrasting in situ hybridization patterns in apomictic and sexual plant ovules from premeiosis to anthesis. Here we transformed sexual P. notatum with a TGS1-like antisense construction under a constitutive promoter, in order to produce lines with reduced transcript representation. Antisense plants developed prominent trichomes on the adaxial leaf surface, a trait absent from control genotypes. Reproductive development analysis revealed occasional formation of twin ovules. While control individuals typically displayed a single meiotic embryo sac per ovule, antisense lines showed 12.93-15.79% of ovules bearing extra nuclei, which can be assigned to aposporous-like embryo sacs (AES-like) or, alternatively, to gametophytes with a misguided cell fate development. Moreover, around 8.42-9.52% of ovules showed what looked like a combination of meiotic and aposporous-like sacs. Besides, 32.5% of ovules at early developmental stages displayed nucellar cells with prominent nuclei resembling apospory initials (AIs), which surrounded the megaspore mother cell (MMC) or the MMC-derived meiotic products. Two or more concurrent meiosis events were never detected, which suggest a non-reduced nature for the extra nuclei observed in the mature ovules, unless they were generated by proliferation and misguided differentiation of the legitimate meiotic products. The antisense lines produced a similar amount of viable even-sized pollen with respect to control genotypes, and formed an equivalent full seed set (∼9% of total seeds) after self-pollination. Flow cytometry analyses of caryopses derived from antisense lines revealed that all full seeds had originated from meiotic embryo sacs (i.e. by sexuality). A reduction of 25.55% in the germination percentage was detected when comparing antisense lines with controls. Our results indicate that PN_ TGS1-like influences ovule, gametophyte and possibly embryo development.

Dataset Information

Structure, target-specificity and expression of PN_LNC_N13, a long non-coding RNA differentially expressed in apomictic and sexual Paspalum notatum.

Publications

Structure, target-specificity and expression of PN_LNC_N13, a long non-coding RNA differentially expressed in apomictic and sexual Paspalum notatum.

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