Sexual and Apogamous Species of Woodferns Show Different Protein and Phytohormone Profiles

Abstract

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.

Keywords: Dryopteris affinis ssp. affinis; Dryopteris oreades; apogamy; apomixis; fern; gametophyte; plant growth regulator; proteomic.

Figure 1

Morphological features in the growth stages of the apogamous fern Dryopteris affinis: (A) filamentous; (B) spatulate; (C) cordate or heart-shaped; (D) detail of first steps in the embryo development; and its sexual relative D. oreades: (E) archegonium cushion in the middle of gametophyte; (F) archegonia.

Figure 2

Venn diagram totaling the number of proteins similarly and differently regulated in heart-shaped gametophytes from the apomictic fern Dryopteris affinis (DA), and its sexual relative D. oreades (DO).

Figure 3

Number of protein clusters obtained from the apogamous gametophytes of Dryopteris affinis and its sexual relative D. oreades, with the best hits to species belonging to the phylogenetic groups specified.

Figure 4

GO classification of proteins differentially upregulated in the apogamous fern Dryopteris affinis and its sexual relative D. oreades, in the three main categories: (A) biological function; (B) molecular function; and (C) cellular component.

Figure 5

KEGG classification of proteins differentially upregulated in the apogamous fern Dryopteris affinis and its sexual relative D. oreades.

Figure 6

PCA biplot showing the contribution of all variables to the variation in the endogenous content of phytohormones in filamentous (F), spatulate (S), and heart-shaped (H) gametophytes of the apogamous species Dryopteris affinis, and heart-shaped gametophytes (O) of its sexual relative D. oreades. Ellipses encircle group samples from the same origin. IAA, indol-3-acetic acid; BA, 6-benzylaminopurine; Z, trans-Zeatin; Zr, Zeatin riboside; DHZ, dihydrozeatin; DHZR, dihydrozeatin riboside; iP, isopentenyl adenine; iPR, isopentenyl adenosine; ABA, abscisic acid; SA, salicylic acid; GA₃, gibberellic acid; GA₄, gibberellin 4; BL, brassinolide; CS, castasterone.