The gametophyte of ferns reproduces either by sexual or asexual means. In the latter, apogamy represents a peculiar case of apomixis, in which an embryo is formed from somatic cells. A proteomic and physiological approach was applied to the apogamous fern Dryopteris affinis ssp. affinis and its sexual relative D. oreades. The proteomic analysis compared apogamous vs. female gametophytes, whereas the phytohormone study included, in addition to females, three apogamous stages (filamentous, spatulate, and cordate). The proteomic profiles revealed a total of 879 proteins and, after annotation, different regulation was found in 206 proteins of D. affinis and 166 of its sexual counterpart. The proteins upregulated in D. affinis are mostly associated to protein metabolism (including folding, transport, and proteolysis), ribosome biogenesis, gene expression and translation, while in the sexual counterpart, they account largely for starch and sucrose metabolism, generation of energy and photosynthesis. Likewise, ultra-performance liquid chromatography-tandem spectrometry (UHPLC-MS/MS) was used to assess the levels of indol-3-acetic acid (IAA); the cytokinins: 6-benzylaminopurine (BA), trans-Zeatine (Z), trans-Zeatin riboside (ZR), dyhidrozeatine (DHZ), dyhidrozeatin riboside (DHZR), isopentenyl adenine (iP), isopentenyl adenosine (iPR), abscisic acid (ABA), the gibberellins GA3 and GA4, salicylic acid (SA), and the brassinosteroids: brassinolide (BL) and castasterone (CS). IAA, the cytokinins Z, ZR, iPR, the gibberellin GA4, the brassinosteoids castasterone, and ABA accumulated more in the sexual gametophyte than in the apogamous one. When comparing the three apogamous stages, BA and SA peaked in filamentous, GA3 and BL in spatulate and DHRZ in cordate gametophytes. The results point to the existence of large metabolic differences between apogamous and sexual gametophytes, and invite to consider the fern gametophyte as a good experimental system to deepen our understanding of plant reproduction.

Apomixis was originally defined as the replacement of sexual reproduction by an asexual process that does not involve fertilization but, in angiosperms, it is often used in the more restricted sense of asexual reproduction through seeds. In ferns, apomixis combines the production of unreduced spores (diplospory) and the formation of sporophytes from somatic cells of the prothallium (apogamy). The genes that control the onset of apogamy in ferns are largely unknown. In this study, we describe the gametophyte transcriptome of the apogamous fern Dryopteris affinis ssp. affinis using an RNA-Seq approach to compare the gene expression profiles of one- and two-dimensional gametophytes, the latter containing apogamic centers. After collapsing highly similar de novo transcripts, we obtained 166,191 unigenes, of which 30% could be annotated using public databases. Multiple quality metrics indicate a good quality of the de novo transcriptome with a low level of fragmentation. Our data show a total of 10,679 genes (6% of all genes) to be differentially expressed between gametophytes of filamentous (one-dimensional) and prothallial (two-dimensional) architecture. 6,110 genes were up-regulated in two-dimensional relative to one-dimensional gametophytes, some of which are implicated in the regulation of meristem growth, auxin signaling, reproduction, and sucrose metabolism. 4,570 genes were down-regulated in two-dimensional versus one-dimensional gametophytes, which are enriched in stimulus and defense genes, as well as genes involved in epigenetic gene regulation and ubiquitin degradation. Our results provide insights into free-living gametophyte development, focusing on the filamentous-to-prothallus growth transition, and provide a useful resource for further investigations of asexual reproduction.