An exonuclease V homologue is expressed predominantly during early megasporogenesis in apomictic Brachiaria brizantha.

MAIN CONCLUSION: An exonuclease V homologue from apomictic Brachiaria brizantha is expressed and localized in nucellar cells at key moments when these cells differentiate to give rise to unreduced gametophytes. Brachiaria is a genus of forage grasses with economical and agricultural importance to Brazil. Brachiaria reproduces by aposporic apomixis, in which unreduced embryo sacs, derived from nucellar cells, other than the megaspore mother cell (MMC), are formed. The unreduced embryo sacs produce an embryo without fertilization resulting in clones of the mother plant. Comparative gene expression analysis in ovaries of sexual and apomictic Brachiaria spp. revealed a sequence from B. brizantha that showed a distinct pattern of expression in ovaries of sexual and apomictic plants. In this work, we describe a gene named BbrizExoV with strong identity to exonuclease V (Exo V) genes from other grasses. Sequence analysis in signal prediction tools showed that BbrizExoV might have dual localization, depending on the translation point. A longer form to the nucleus and a shorter form which would be directed to the chloroplast. This is also the case for monocot sequences analyzed from other species. The long form of BbrizExoV protein localizes to the nucleus of onion epidermal cells. Analysis of ExoV proteins from dicot species, with exception of Arabidopsis thaliana ExoVL protein, showed only one localization. Using a template-based AlphaFold 2 modelling approach the structure of BbrizExoV in complex with metal and ssDNA was predicted based on the holo structure of the human counterpart. Features predicted to define ssDNA binding but a lack of sequence specificity are shared between the human enzyme and BbrizExoV. Expression analyses indicated the precise site and timing of transcript accumulation during ovule development, which coincides with the differentiation of nucelar cells to form the typical aposporic four-celled unreduced gametophyte. A putative function for this protein is proposed based on its homology and expression pattern.

Toward in vitro fertilization in Brachiaria spp.

Brachiaria are forage grasses widely cultivated in tropical areas. In vitro pollination was applied to accessions of Brachiaria spp. by placing pollen of non-dehiscent anthers on a solid medium near isolated ovaries. Viability and in vitro germination were tested in order to establish good conditions for pollen development. Comparing sexual to apomictic plants, apomictic pollen has more abortion after meiosis during the microspore stage and a lower viability and, of both types, only some plants have sufficient germination in a high sugar concentration. Using in vitro pollination with the sexual plant, the pollen tube penetrates into the nucellus and micropyle, but the embryo sac degenerates and collapses. In the apomictic B. decumbens, in vitro pollination leads to the transfer of the sperm nuclei into the egg cell and the central cell. The results are discussed according to normal fertilization and barriers in sexual and apomictic plants.