An even more unpredictable result was that the labelling of starch with anti-AGP antibodies was different according to the starch grain type

An even more unpredictable result was that the labelling of starch with anti-AGP antibodies was different according to the starch grain type. reproductive cycle (Coimbra (Hydatellaceae), which is the closest extant relative of the water-lilies (Saarela species represent useful models for comparative studies of this type, because (uniquely for early-divergent angiosperms) they are mostly rapidly growing annual plants that are readily cultivated grown at the Royal Botanic Gardens, Kew, were fixed in 2 % paraformaldehyde and 25 %25 % glutaraldehyde in phosphate buffer (0025 m, pH 7, with one micro drop of Tween 80), placed under vacuum for 1 h and then at 4 C overnight. After dehydration in a graded ethanol series, the material was embedded in LR White embedding resin (London Resin Company Ltd, London, UK). Thick sections (05 m) were obtained with a Leica Reichert Supernova microtome placed on glass slides, and stained with a solution of 1 1 % methylene blue for light microscopy, and 1 % Lugol solution (Sigma-Aldrich 62650; St Louis, MO, USA) for starch staining. Some slides were preserved unstained for immunolocalization of AGPs and pectin epitopes with monoclonal antibodies. Immunolocalization of AGPs and pectins A collection of monoclonal antibodies directed against glycosyl moieties speci? c to AGPs and pectins was provided by Prof. Paul Knox from the Centre for Plant Sciences, Epertinib hydrochloride Faculty of Biological Sciences and University of Leeds, UK. The monoclonal antibodies recognizing AGP epitopes used were JIM8 (Pennell typically consist of numerous pistils surrounding one or two central stamens (Fig.?1A), all the fertile organs being enclosed by an involucre of bract-like phyllomes that resemble perianth organs. Pre-fertilized carpels were characterized here, which already contain the main seed storage tissue, Epertinib hydrochloride the perisperm (e.g. Rudall (2009reproductive unit, with a single stamen in the centre (labelled A) surrounded by several pistils, each with several long, multicellular uniseriate stigmatic hairs (arrows); scale bar = Epertinib hydrochloride 200 m. (B) Detail of lower part of ovule and carpel wall showing perisperm (PS) (*) with aggregated nuclei and two integuments, each with two cell layers (arrow, inner integument; arrowhead, outer integument); scale bar = 50 m. (C) Detail of upper part of ovule and carpel wall showing the nucellar beak; scale bar = 50 m. Immunolocalization of AGPs and pectins in developing seeds of some AGPs were identified as molecular markers for gametophytic cell development (Coimbra were differentially labelled by very similar anti-AGP antibodies such as JIM8 and JIM13. Specifically, JIM8 and LM2 both labelled equally; they are both absent from the perisperm starch grains (Fig.?4B, C), but are present in the outer layer of the starch grains of the outer integument, ovary wall, stamen filaments and bract-like phyllomes (Fig.?4B, E, F). Conversely, JIM13-recognized epitopes were present in the outer layer of the perisperm starch grains (Fig.?4A, D) and also present in the starch grains of the ovary wall, stamen filaments and bract-like phyllomes (Fig.?4A, G). In these plants, the GLUR3 amyloplasts may contain one or more starch grains; several grains of joint origin form a compound amyloplast, as evidenced by staining with lugol (Fig.?4H, I). Open in a separate window Fig. 4. Starch grains of different tissues in the same reproductive unit. (A) JIM13 labels the starch grains of the perisperm (*) and the outer integument (arrow). (B) JIM 8 and (C) LM2 both label only the Epertinib hydrochloride starch grains present in the outer integument and ovary wall (arrows) and not the perisperm starch grains. (D) The labelling is associated with the outer layer of the perisperm starch grains. (E) JIM 8 and (F) LM2 both label equally the outer layer.