The Role of APOSTART in Switching between Sexuality and Apomixis in Poa pratensis

Abstract

The production of seeds without sex is considered the holy grail of plant biology. The transfer of apomixis to various crop species has the potential to transform plant breeding, since it will allow new varieties to retain valuable traits thorough asexual reproduction. Therefore, a greater molecular understanding of apomixis is fundamental. In a previous work we identified a gene, namely APOSTART, that seemed to be involved in this asexual mode of reproduction, which is very common in Poa pratensis L., and here we present a detailed work aimed at clarifying its role in apomixis. In situ hybridization showed that PpAPOSTART is expressed in reproductive tissues from pre-meiosis to embryo development. Interestingly, it is expressed early in few nucellar cells of apomictic individuals possibly switching from a somatic to a reproductive cell as in aposporic apomixis. Moreover, out of 13 APOSTART members, we identified one, APOSTART_6, as specifically expressed in flower tissue. APOSTART_6 also exhibited delayed expression in apomictic genotypes when compared with sexual types. Most importantly, the SCAR (Sequence Characterized Amplified Region) derived from the APOSTART_6 sequence completely co-segregated with apomixis.

Keywords: APOSTART; Poa pratensis; apomixis; plant reproduction.

Figure 1

qPCR (quantitative PCR) profile of six APOSTARTs in two different sets of genotypes of P. pratensis (orange, apomictic genotypes; blue, sexual genotypes). The expression level was evaluated in five different flowering stages (pre-meiosis; meiosis; post-meiosis; anthesis; post-anthesis) and two tissues (leaves and roots).

Figure 2

qPCR profile of APOSTART_6 member in three different sets of genotypes (orange, apomictic genotypes; blue, sexual genotypes; purple, parthenogenetic recombinant genotype).

Figure 3

(A) APO-SCAR primer pair tested on 48 F1 individuals (lanes 1–48) from a segregating population for the mode of reproduction and obtained by crossing an apomictic (RS7, lane 50) and a sexual (S1/1, lane 51) genotype, as reported in Albertini et al. 2001 [12]. APO-SCAR completely co-segregate with apomixis; (B) on exotic germplasm sources of known reproductive behavior (Table S1): lanes 1–5 and 11, apomictic PaPp F₁ hybrids; lane 6, sexual P. arachnifera maternal parent (Pa1FM); lane 7, apomictic KB3 P. pratensis pollen parent; lanes 8–10, non-apomictic PaPp F₁ hybrids; lanes 12–13, apomictic 9601, 1915 P. pratensis.

Figure 4

Maximum-likelihood phylogenetic unrooted tree based on APOSTART_6 similar amino acid sequences. The tree has the highest log likelihood (-45391.1656) and is condensed with bootstrap value >70. Phylogenetic analysis was performed using Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model and a discrete γ distribution (5 categories (+G, parameter = 0.6937)). The analysis involved 208 amino acid sequences and was conducted in MEGA7 [23]. Bootstrap values are reported in Figure S3 for more accuracy. Poaceae are indicated in red, Solanaceae in light blue, Asteraceae in green, Malvaceae in yellow, Brassicaceae in purple, Rosaceae in black, and Fagaceae in dark blue.

Figure 5

APOSTART expression in longitudinal sections of flowers studied by ISH. Sections of sexual and apomictic genotypes were probed with DIG-labeled antisense (B,C,E,F,H,I,K,L,N,O) or sense (A,D,G,J,M) RNAs and viewed under a microscope bright field that gives a purple label. No signals were detected in the sections hybridized with the sense probes ((A,J,M) sexual genotypes; (D,G) apomictic genotypes). (B,C), longitudinal sections of ovules of sexual (B) and apomictic (C) genotypes containing the MMC, in both genotypes a poor hybridization signal was observed. In sexual (E) and apomictic (F) genotype ovules during megasporogenesis, the hybridization signal detected in the MMC was the same background in both genotypes, while in apomictic ovules a strong signal was observed in the aposporic cells (indicated by a black arrow) near the MMC (F). (H,I), dyads and tetrads observed in ovules of sexual (H) and apomictic (I) genotypes have the same signal the background unless they are destined to degenerate. (K,L), at the anthesis the hybridization signal was strong in the sexual embryo sac (K) and stronger in the apomictic embryo sacs (L). (N,O), a strong hybridization signal was observed also in the embryo of sexual (N) and apomictic (O) genotypes. a = anther; d = dyad; e = embryo; ed = endosperm; es = embryo sac; mmc = megaspore mother cell; t = tetrad. Bars = 40 μm.

Figure 6

Consensus scoring of stigmasterol (black), brassicasterol (dark gray), and campesterol (light gray) poses binding at APOSTART_1 (left), APOSTART_6 (middle), and APOSTART_8 (right) obtained after 500 ps of MD (Molecular Dynamics) simulation (vide infra).

Figure 7

Representation of the binding complexes of APOSTART_1 and APOSTART_6 (cartoon) with the considered phytosterols (sticks). Binding sites of stigmasterol (green), brassicasterol (gray), and campesterol (orange) at the two proteins are also displayed.