Functional characterization and role of PnMEKK1 in aposporous development.

MANCINI M; PERMINGEAT H; PODIO M; SIENA LA; PUPILLI F; DEMARCHI L; GALUPPO F; ARRAIS-GUIMARAES L; DUSI D; TAVARES DE CAMPOS CARNEIRO V; FELITTI S; BELTRÁN C; SARTOR M; SEIJO G; GONZÁLEZ AM; ORTIZ JPA; PESSINO SC

Bahía Blanca

Workshop; IV Ciclo de seminarios sobre avances en la caracterización genética y molecular de la apomixis en gramíneas forrajeras; 2014

CERZOS-CONICET

Apomixis is an asexual mode of plant reproduction through seeds. After using differential display analysis to compare the floral transcriptome of sexual and apomictic Paspalum notatum plants, we identified an mRNA transcript fragment (N46) similar to genes encoding mitogen-activated protein kinase kinase kinases. Three to four copies representing alleles/paralogues were detected by using genomic Southern hybridization. Next generation long-read sequencing revealed only one splice variant (isotig) expressed in flowers of the sexual plant, but four in the apomictic one. Since strong similarity to MAP3Ks/MEKKs was confirmed, the gene was renamed 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 premeiosismeiosis, but down-regulated in sexual plants. Biolistic transformation was used to co-introduce two plasmid constructs, including a PnMEKK1 hairpin cloned under the rice ACT1 promoter and the reporter EGFP under the maize UBI promoter, 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 a component of a molecular cascade expressed in the nucellus of aposporous plants, which is involved in the formation of non-reduced embryo sacs, but not in the occurrence of the sexual reproductive pathway.