Figure 1.
PIN1 and PIN3 expression in ovules.
A–F, PIN1; G–I PIN3 A–D CLSM analysis on PIN1:PIN1-GFP lines.At stage 1-I PIN1 is basal polar localized in the nucellar cells shortly after the formation of the ovule primordia (A). At stage 2–II, PIN1 is expressed in the nucellus and in the inner integument primordia (B). Closer view of a nucellus (ovule stage 2–V) (C). In developing ovules at stage 3-II, PIN1 is still basal polar localized (D). (E–F) PIN1 immuno-localisation experiments with an anti-PIN1 antibody. PIN1 is already detectable in the ovule primordium. (E) ovule primordia, stage 1–I; (F) ovule at stage 2–I. (G–I) CLSM analysis of PIN3:PIN3-GFP. PIN3 protein is expressed in the nucellus since stage 1–II (G) and its expression persists until stage 3–V (I). PIN3 is also expressed in the funiculus vasculature throughout ovule development, in the insert a closer view of the funiculus vasculature (I). In (J) a summary of PIN1 and PIN3 localisation in developing ovules. Ovule developmental stages are labelled following the convention of Schneitz and co-workers [1]. fg, female gametophyte; ii, inner integument; oi, outer integument; fun, funiculus; nu, nucellus. Scale bars: 20 µm.
Figure 2.
The Auxin synthesis and accumulation in wild type ovules.
Wild-type DR5rev:GFP (A–E) and DR5rev:3XVENUS-N7 (F) ovules were analysed at different developmental stages. The cytoplasmatic GFP signal is first detected in the nucellus of the ovule primordium from stage 1–II (A) until stage 3–III (D). DR5rev:GFP signal is also detected in the forming ovule vasculature (asterisks in C and E). (F) the florescent signal confirms that the auxin response is only detected in the maternal nucellar cells, the YFP is fused to a nuclear localization sequence. In A–D cell membranes were stained with FM® 4–64 FX. Wild-type TAA1:GFP expression pattern in developing ovules at stages 2–I (G), 2–III (H), and 3–II (I). (J) YUC4:GUS is expressed since stage 3–II in the inner integument cells. The YUC4 promoter is active in the inner integument in the cells close to the micropyle starting from stage 3–II. fg, female gametophyte; ii, inner integument; oi, outer integument; fun, funiculus; nu, nucellus Scale bars: 20 µm.
Figure 3.
PIN1 down regulation affects ovule development.
(A and B) pin1-5 ovules with defective female gametophyte. The female gametophytes arrest in development at FG1. (C and D) ovules of the PIN1 down-regulation (pDEFH9:amiPIN1) plants show female gametophyte defects. Female gametophytes are blocked at stages FG1 and FG3. (E) mature wild-type ovules, with FG7 female gametophyte. (F) Real-time PCR to quantify PIN1 and PIN3 expression levels in transgenic plants expressing the artificial microRNA against PIN1 and PIN3 (light grey transgenic plants showing ovule abortion, dark grey sibling wild-type plants with normal seed set). Expression of the artificial microRNA is under the control of the ovule specific pDEFH9 promoter. Scale bars: 20 µm nu, nucellus; fun, funiculus; ii, inner integument; oi, outer integument; fg, female gametophyte.
Figure 4.
Model of auxin flux and production during ovule development.
The diagram summarises the auxin response (in green), auxin flux (red arrows) and auxin synthesis (grey) in developing ovules from stage 2–I (A) to stage 3–IV (D). At stage 2–I (A) the megaspore mother cell (MMC) is recognisable. At this stage the auxin response (DR5rev::GFP) is restricted to a few cells of the nucellus; PIN1 is also detected in the nucellar cells and has is a basal polar localization. At this stage pTAA1 drives reporter expression in the ovule chalaza region and in the funiculus. At stage 2–III (B) PIN1 is detected in the inner integument primordia and in the funiculus, comcometeley with the auxin response. At stage 2–IV (C) the TAA1 promoter reduces its expression in the inner integument but at the tip of the inner integument YUC4 begins to be expressed. At this stage PIN1 and the auxin response are restricted to the funiculus since the nucellus is degenerating. The position of PIN1 in the nucellus cells strongly indicates its importance in auxin exit from these cells. The exit of auxin stimulates megagametogenesis as shown by PIN1 silencing. fg, female gametophyte; ii, inner integument; oi, outer integument; fun, funiculus; chal, chalaza.