Extended Data Fig. 1: FER-regulated pollen tube–ovule interaction and pectin deposition in the filiform apparatus.
From: FERONIA controls pectin- and nitric oxide-mediated male–female interaction
a, Blue-dot assay, an alternate method (from that in Fig. 1b) to show pollen tube targeting and penetration of ovules. β-GUS-expressing pollen grains (Pg, blue) were used to pollinate a wild-type pistil, allowing the pollen tube growth path to be visualized and demonstrated the prevalently 1:1 pollen tube:ovule ratio under normal pollination conditions. This result is typical of pollinated wild-type pistils, and it was repeated at least five times in the course of this study. b, FER promoter (FERp)::FER–GFP expression in a complemented fer-4 pistil, showing prominent localization of FER–GFP at the filiform apparatus region (arrows)4 as well as expression in sporophytic tissues, such as the integuments (In) and funiculus (F). For this study, the observation was repeated at least three times in independent preparations. c, Pollen tube reception defects induced by fer-4 mutation (pollen tube overgrowth inside the female gametophyte owing to non-rupture) and multiple pollen tube entrance phenotypes. d–f, RuR-detected pectin in ovules. d, Wild-type and fer-4 ovules showed developmental regulation and FER-dependent pectin deposition at the filiform apparatus or micropylar region (arrowheads). Floral stages followed that of previous publications4,46; stages 13–15 are the most receptive for pollination—at stage 16 ovules had enlarged, reflecting successful fertilization. e, Quantification of RuR-positive filiform apparatus region. f, FERp::FER–GFP complemented fer-4 mutation restored deposition of de-esterified pectin at the filiform apparatus, as it did the normal single pollen tube penetration phenotype (Extended Data Fig. 1c). g, Quantification of de-esterified pectin located at the filiform apparatus by JIM5 and M38 signal levels, following a previously described method4. Average signal intensity in equal areas of interest (dotted lines) at the filiform apparatus and in the synergid cells were compared. A filiform apparatus:synergid cell ratio of ≥1.2 was scored as JIM5- or M38-positive. h, JIM7 immunostaining of ovule methylesterified pectin. Arrows, filiform apparatus region. Image acquisition conditions were the same as those for JIM5 immunostaining (Fig. 1e). Signal was below detection, most probably because of the high solubility of the methylesterified polymers during the immunostaining procedure, which involved extensive washes. i, JIM5 immunostaining of wild-type and FERp::PMEI1–GFP (PMEI1ox) ovules. Overexpression of PMEI1 (Extended Data Fig. 2) reduced the localization of de-esterified pectin at the filiform apparatus. j, M38 immunostaining of wild-type, pme44, pme34 and PMEIox ovules. Reduced M38 and JIM5 signals correlated with augmented multiple pollen tube entry phenotype (Fig. 1g). Data shown are average ± s.d. n, pistil numbers (c, e, f, h–j); n, ovule numbers (g). Data are representative of three independent experiments. P values were obtained by two-tailed t-tests; numbers in data plots represent the number of ovules examined. Scale bar, 1 mm (a), 50 μm (d, g, h). Box plots: centre line, median; box limits, lower and upper quartiles; dots, individual data points; whiskers, highest and lowest data points.
