IICAR   25568
INSTITUTO DE INVESTIGACIONES EN CIENCIAS AGRARIAS DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Exploring the link between TGS1-like downregulation and the transition from sexuality to apomixis in Paspalum notatum
Autor/es:
COLONO, CAROLINA; LEBLANC, OLIVIER; SIENA, LORENA; PERMINGEAT, HUGO; ORTIZ, JUAN PABLO AMELIO; PESSINO, SILVINA
Lugar:
Bahía Blanca
Reunión:
Workshop; V Ciclo de seminarios sobre avances en la caracterización genética y molecular de la apomixis; 2016
Institución organizadora:
CERZOS-CONICET
Resumen:
Apomixis (asexual reproduction via seeds) is an odd developmental process leading to the formation of a clonal offspring genetically identical to the mother plant, in absence of meiosis and fertilization. It is conceived as a deviation from the sexual reproductive route, caused by the emergence of variants of one to several genetic/epigenetic components. In prior work, we discovered that a paralog of gene TGS1 (trimethyl guanosyl synthase-1), named TGS1-like, was differentially expressed in ovules of sexual and apomictic Paspalum notatum plants from premeiosis to anthesis. The expression of TGS1-like was downregulated in apomicts, and displayed a positive correlation with the sexuality rate. While TGS1 encodes a short protein (~450 aa) with a methyltransferase domain, TGS1-like is longer (~540 aa) and display an extended N-terminal domain including a WW protein binding site. In yeast and animals, the TGS1 protein has a dual role: 1) promotes the completion of sn(o)RNAs biogenesis, by catalyzing the post-transcriptional conversion of 7-methylguanosine caps (m7G) into 2,2,7-trimethylguanosine (m3G), a process essential to mRNAs splicing, rRNA processing and telomerase activity; 2) acts as a PRIP (peroxisome proliferator-activated receptor-interacting protein)-associated coactivator in transcription mediator complexes. In eukaryotic non-plant model systems, the abolishment of TGS1 function causes pre-rRNA processing deficiency, loss of nucleolar structural organization, and aberrant splicing of key regulators. This originates a wide range of phenotypic alterations, including cold-sensitive growth defects and meiosis failure. On the contrary, the function of TGS1-like was not characterized yet. Our objective was to study the possible role of TGS1-like in reproductive development. Based on the set of activities described in other taxa for its paralog, TGS1, we hypothesized that apomicts plants produce specific splice variants in flowers, which are concurrent with TGS1-like down-regulation. To put this hypothesis to the test, we characterized splice variants occurring in florets of sexual and apomictic P. notatum plants. A list of candidate genes with differential representation in 454/Roche floral transcriptome libraries was examined. We selected 120 candidates showing the lowest probability values for identical expression. Bioinformatic analyses indicated that 17 were possibly represented by different splice variants. Three transcripts (>10779, >22630 and >23387) were selected to be studied in a larger number of apomictic and sexual individuals. Intron-specific oligonucleotides were designed and used in RT-PCR and qPCR experiments to detect the unprocessed isotigs. For transcripts >10779 and >23387, alternative splicing was confirmed. Moreover, >23387 differential splicing was linked to the reproductive mode in 4 apomictic and 4 sexual plants. Isotig >23387 is the Paspalum ortholog to Arabidopsis gene LHCA1, encoding a protein that function as a membrane attachment module. The gene was associated with asymmetric division developmental pathways operating in stomata, trichomes and root epidermis cells; it is expressed in all these locations as well as in leaves, ovules and immature embryos. The splice variant differentially expressed in sexual and apomictic Paspalum plants is analogous to Arabidopsis LHCA1.4. In order to confirm a cause-effect link between TGS1-like activity and the occurrence of LHCA1.4 as well as other specific splice variants, we decided to investigate P. notatum sexual plants transformed with a TGS1-like antisense construction. A 733 bp specific CDR region (F1 fragment) was cloned in antisense orientation within vector pAct1-gfbsd2 (pAct1-F1as). Undifferentiated callus induced from mature seeds of sexual P. notatum were co-transformed with particles carrying pAct1-F1as + pAct1-GFPBAR (containing reporter GFP and selector BAR), by using a Biomics gene gun. Two experiments involving 10 plates with 15 calluses each (and including selection and regeneration controls) were conducted. GFP fluorescence is now being controlled in roots using a Nikon E200 light transmission microscope. We plan to analyze PNLHCA1 splice variants? representation levels by performing qPCR experiments in transformant and control plants. Moreover, we will examine alterations in reproductive development by using DIC microscopy. Similar explorations are being conducted in Arabidopsis tgs1-like defective mutants.