AGPs as molecular determinants of reproductive development

Abstract

Background and aims: Morphogenesis occurs through accurate interaction between essential players to generate highly specialized plant organs. Fruit structure and function are triggered by a neat transcriptional control involving distinct regulator genes encoding transcription factors (TFs) or signalling proteins, such as the C2H2/C2HC zinc-finger NO TRANSMITTING TRACT (NTT) or the MADS-box protein SEEDSTICK (STK), which are important in setting plant reproductive competence, feasibly by affecting cell wall polysaccharide and lipid distribution. Arabinogalactan proteins (AGPs) are major components of the cell wall and are thought to be involved in the reproductive process as important players in specific stages of development. The detection of AGPs epitopes in reproductive tissues of NTT and other fruit development-related TFs, such as MADS-box proteins including SHATTERPROOF1 (SHP1), SHP2 and STK, was the focus of this study.

Methods: We used fluorescence microscopy to perform immunolocalization analyses on stk and ntt single mutants, on the ntt stk double mutant and on the stk shp1 shp2 triple mutant using specific anti-AGP monoclonal antibodies. In these mutants, the expression levels of selected AGP genes were also measured by quantitative real-time PCR and compared with the respective expression in wild-type (WT) plants.

Key results: The present immunolocalization study collects information on the distribution patterns of specific AGPs in Arabidopsis female reproductive tissues, complemented by the quantification of AGP expression levels, comparing WT, stk and ntt single mutants, the ntt stk double mutant and the stk shp1 shp2 triple mutant.

Conclusions: These findings reveal distinct AGP distribution patterns in different developmental mutants related to the female reproductive unit in Arabidopsis. The value of the immunofluorescence labelling technique is highlighted in this study as an invaluable tool to dissect the remodelling nature of the cell wall in developmental processes.

Keywords: NO TRANSMITTING TRACT; SEEDSTICK; SHATTERPROOF; Sexual plant reproduction; arabinogalactan proteins; immunolocalization.

Fig. 1.

Immunolocalization of AGPs in Arabidopsis flower mutants with the monoclonal antibody JIM8 by fluorescence microscopy: WT (A, B); ntt single mutant (C, D); stk single mutant (E, F); ntt stk double mutant (G, H); and stk shp1 shp2 triple mutant (I, J). Abbreviations: es, embryo sac; f, funiculus; fa, filiform apparatus; fvb, funiculus vascular bundles; ii, internal integument; l, locule; m, micropylar region; o, ovule; ow, ovary wall; r, replum; s, seed; sp, septum; sy, synergids; *, exudate. Flowers were collected at stages 14–15 of flower development according to Smyth et al. (1990). Scale bars: 20 µm.

Fig. 2.

Immunolocalization of AGPs in Arabidopsis flower mutants with the monoclonal antibody JIM13 by fluorescence microscopy: WT (A, B); ntt single mutant (C); stk single mutant (D, E); ntt stk double mutant (F–H); and stk shp1 shp2 triple mutant (I, J). Abbreviations: e, endosperm; es, embryo sac; f, funiculus; fa, filiform apparatus; fvb, funiculus vascular bundles; ii, internal integument; l, locule; lv, lateral vascular bundles; m, micropylar region; mv, medial vascular bundles; ow, ovary wall; r, replum; s, seed; sp; septum; sy, synergids; *, exudate. Flowers were collected at stages 14–15 of flower development according to Smyth et al. (1990). Scale bars: 20 µm (A–E, G–J); 100 µm (F).

Fig. 3.

Immunolocalization of AGPs in Arabidopsis flower mutants with the monoclonal antibody CCRC-M31 by fluorescence microscopy: WT (A, B); stk single mutant (C); stk single mutant close-up view (D); ntt single mutant (E); ntt stk double mutant (F, G); and stk shp1 shp2 triple mutant (H–J). Abbreviations: cc, central cell; es, embryo sac; f, funiculus; fa, filiform apparatus; ii, internal integument; l, locule; lv, lateral vascular bundles; m, micropylar region; o, ovule; ow, ovary wall; r, replum; s, seed; sp, septum; sy, synergids; tt, transmitting tract. Flowers were collected at stages 14–15 of flower development according to Smyth et al. (1990). Scale bars: 20 µm (A, B, D–F, G–J); 100 µm (C).

Fig. 4.

Quantitative PCR relative expression levels of the selected AGP mRNA transcripts in inflorescences of the WT, stk and ntt single mutants, ntt stk double mutant and stk shp1 shp2 triple mutant in Arabidopsis plants. The level of the transcripts was normalized using the reference genes ACT8 and RUB1. Each bar represents an average of the three technical replicates. *Statistically significant result (P < 0.05).

Fig. 5.

Diagram showing the spatiotemporal distribution of AGPs epitopes (JIM8, JIM13 and CCRC-M31) throughout the female reproductive structures and tissues in the WT (A) compared with the ntt (B) and stk (C) single mutants, the ntt stk double mutant (D) and the stk shp1 shp2 triple mutant (E) in Arabidopsis plants. Areas in grey represent no labelling significance. Abbreviations: es, embryo sac; f, funiculus; m, micropylar region; ow, ovary wall; tt, transmitting tract.