Extended Data Fig. 2: Characterization of mutants deficient in de-esterified pectin.
From: FERONIA controls pectin- and nitric oxide-mediated male–female interaction
a–d, Transfer (T-) DNA insertion mutants pme34-1 (Salk_062058)30 and pme44-1 (SALK_071362). a, c, Genomic maps with T-DNA insertions (triangles). b, d, PCR with reverse transcription for PME34 and PME44 mRNAs from seedlings (S) and flowers (F). Green arrows, primers for RT–PCR; black and red arrow pairs, primers for genotyping. e–g, Flowering wild-type, pme34-1, pme44-1 and PMEI1ox plants. Growth, flowering time, flower morphology and reproductive yields (silique sizes, arrows in f, g, and seed numbers) of these transgenic mutant plants were adequately normal relative to wild type, permitting reproductive studies. h–k, FERp::PMEI1–GFP expression and ovule morphology in PMEI1ox plants. PMEI1–GFP expression in seedlings and ovules paralleled that of FERp::FER–GFP4,10, including a most-prominent accumulation in the elongation zone of seedling roots (h), throughout the ovules and prominently at the filiform apparatus region (arrowheads) (i). Ovules appeared mostly normal, although higher level of autofluorescence was observed in the sporophytic tissue of some ovules (white arrows). When stained by aniline blue, most ovules from the plants used here appeared normal (j). Some ovules showed elevated callose deposition in the sporophytic as well as in the female gametophyte region (k), reflecting stress. Ovules with overaccumulation of callose are generally not penetrated by pollen tubes, so were excluded from these studies. Extended Data Figure 10a–c shows defects in a PMEI5-overexpression line20, precluding its use in these studies. l, Statistical comparison among mutants deficient in de-esterified pectin and transgenic plants. The levels of ovules penetrated by multiple pollen tubes in pme34, pme44 and PMEI1ox plants were not significantly different among them (bracket 1). Deficiency in de-esterified pectin among these mutant ovules was also not significantly different (Extended Data Fig. 1j). Attempts to compound their effect in a pme34 pme44 double mutant (arrowheads indicate a penetrating pollen tube doublet) did not result in a significantly higher multiple-tube penetration phenotype than the single mutant parents (bracket 2). Data for pme34, pme44 and PMEI1ox plants were from Fig. 1g. Data for pme44 and pme34 are average ± s.d. n, number of pistils. Results were representative of three independent experiments. P values were obtained by two-tailed t-tests; numbers in data plots denote the number of ovules examined. Box plots: centre line, median; box limits, lower and upper quartiles; dots, individual data points; whiskers, highest and lowest data points.
