Functional role of candidate pnmekk1 in aposporous development.

MANCINI, M.; PERMINGEAT, H.; PODIO, M.; SIENA, L.A.; PUPILLI, F.; DEMARCHI, L.; GALUPPO, F.; ARRAIS-GUIMARAES, L.; DUSI, D.; CAMPOS CARNEIRO, V.; FELITTI, S.A.; BELTRÁN, C.; SARTOR, M.; SEIJO, G.; GONZÁLEZ, A.M.; ORTIZ, J.P.A.; PESSINO, S.C.

Bahía Blanca

Seminario; IV Ciclo de seminarios sobre avances en la caracterización genética y molecular de la apomixis.; 2014

Departamento de Agronomía de la Universidad Nacional del Sur y el CERZOS-CONICET, Bahía Blanca.

Apomixis is an asexual mode of plant reproduction through seeds, which was described in more than 400 angiosperm species. After using differential display analysis to compare the floral transcriptome of sexual and apomictic plants, we identified an mRNA transcript fragment (N46) similar to genes encoding mitogen-activated protein kinase kinase kinases. Three to four copies probably representing alleles/paralogues were detected through genomic hybridization analysis. The complete coding region of the single allele expressed in flowers was characterized by next generation long-read sequencing. Since strong similarity to MAP3Ks/MEKKs was confirmed, the gene was re-named PNMEKK1. Differential expression validation was achieved by using real time PCR and ISH in ovules of Paspalum notatum and Brachiaria brizantha. The coding strand of the transcript was upregulated in the nucellus of aposporous plants at premeiosis-meiosis, but down-regulated in sexual plants. Biolistic transformation was used to co-introduce two plasmid constructs (including a PNMEKK1 hairpin and the reporter EGFP, respectively) into the obligate aposporous genotype Q4117. Co-transformant plants #29 and #65 (EGFP+/PNMEKK1hairpin+) and transformation controls #51 and #57 (EGFP+/PNMEKK1hairpin-) achieved flowering within the first year, allowing evaluation of reproductive phenotypes. Proportions of viable pollen did not differ significantly among co-transformed plants (#29 and #65), transformation controls (#51 and #57) and the wild-type plant. Conversely, plants #29 and #65 displayed a significantly lower average number of Aposporous Embryo Sacs (AES) per ovule (0.97 and 1.14, respectively), in comparison with the wild-type plant (3.34) as well as the transformation controls #51 and #57 (3.38 and 5.28, respectively). The AES/Meiotic Embryo sacs (MES) ratio was different in plants #29 and #65 with respect to wild-type plant Q4117, with an associated probability of 97.2% and 90.5%, respectively, as indicated by χ2 homogeneity tests. Conversely, the average number of AES per ovule and the ratio AES/MES in control plants #51 and #57 were not different from those of Q4117. The proportion of ovules containing MES was similar among all analysed plants. These results indicated that the introduction of the PNMEKK1 hairpin construction impaired the formation of non-reduced embryo sacs in genotype Q4117, without altering the meiotic embryo sacs representation or decreasing pollen viability. Our findings suggest that PNMEKK1 is component of a molecular cascade expressed in the nucella of aposporous plants, which is involved in the formation of non-reduced embryo sacs, but does not participate in the sexual reproductive pathway.