Expression and functional analysis of candidate genes related to aposporous apomixis in grass species

PODIO, M.; MANCINI, M.; PERMINGEAT, H.; SIENA, L.A.; DELGADO, L.; FELITTI, S.A.; SARTOR, M.; ESPINOZA, F.; ARRAIS-GUIMARAES, L.; DUSI, D.; TAVARES, V.; PUPILLI, F.; LEBLANC, O.; ORTIZ, J.P.A.; PESSINO, S.C.

Oslo

Conferencia; Harnessing Plant Reproduction for Crop Improvement; 2013

European Frontiers of Plant Reproduction Research

In the past decade, comparative transcriptome surveys carried out in Paspalum andBrachiaria sp. provided a vast list of genes displaying differential expression in reproductiveorgans of sexual and apomictic individuals. A candidates?s subset was then selected forfurther characterisation using two main criteria: i) concurrent detection in comparativetranscriptome surveys from both species; and/or ii) occurrence of experimental or in silicopositional linkage to genomic regions associated with apospory control. Here, we reportdetailed expression analyses for Pnserk (somatic embryogenesis receptor kinase), Pnexs(exs domain-containing protein), Pn19 (unknown), Pn46 (MAP3K), Pn108 (far1 domaincontainingprotein), Pn69 (DNA methyltransferase) and PnMTA-70 like (RNAmethyltransferase). Paspalum and Brachiaria candidates? full sequences were obtained byperforming RACE (Rapid amplification of cDNA ends) or Illumina sequencing, respectively.Chronological quantitative expression was analysed by real time PCR in reproductive organsof sexual and apomictic genotypes at pre-meiosis, meiosis, post-meiosis and anthesis.Moreover, in situ hybridization was used to determine more accurately the cell types whereexpression was located. The selected candidates showed contrasting chronological andspatial expression patterns in sexual and apomictic plants, sometimes involving both codingand non-coding strands. Most of them displayed strong sense expression in the nucellus ofaposporous plants. A stable transformation platform is currently being used to explore theirpotential functional role in reproductive development by altering timely or spatially theirexpression pattern. Expression of candidate n46 (MAP3K) was down-regulated in a naturalaposporous genotype via the introduction of a hairpin construct. Transformed plants showeda diminished capacity to form aposporous embryo sacs with respect to wild-type plants andtransformation controls. Collectively, our results suggest the existence of a molecularpathway ectopically activated in the nucellus of aposporous genotypes, which regulates theonset of apospory initials and their commitment to a gametophytic fate.