Volume 25, Issue 3, Pages 310-316 (May 2013) Ethylene Signaling Is Required for Synergid Degeneration and the Establishment of a Pollen Tube Block  Ronny Völz, Juliane Heydlauff, Dagmar Ripper, Ludwig von Lyncker, Rita Groß-Hardt  Developmental Cell  Volume 25, Issue 3, Pages 310-316 (May 2013) DOI: 10.1016/j.devcel.2013.04.001 Copyright © 2013 Elsevier Inc. Terms and Conditions

Developmental Cell 2013 25, 310-316DOI: (10.1016/j.devcel.2013.04.001) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 ein3 eil1 Mutants Fail to Establish a Pollen Tube Block (A and B) CLSM of female wild-type (A) and ein3 eil1 (B) gametophytes. (C) Frequency of mature female gametophytes: wild-type (n = 142) and ein3 eil1 (n = 138). (D and E) Expression of FGR 7.0 in wild-type (D) and ein3 eil1 (E) female gametophytes. (F) Frequency of FGR 7.0 expression: wild-type (n = 323), ein3 eil1 (n = 374). (G–K) Cleared whole mounts of wild-type (G and J) and ein3 eil1 (H and K) seeds and frequency of ovules containing supernumerary pollen tubes (I): wild-type (n = 245) and ein3 eil1 (n = 274). (L) Frequency of synergid-containing seeds analyzed at the four-nucleate endosperm stage: wild-type × wild-type (n = 156), ein3 eil1 × wild-type (n = 157), wild-type × ein3 eil1 (n = 70), and ein3 eil1 × ein3 eil1 (n = 92). (M and N) Expression of FGR 7.0 in wild-type (M) and ein3 eil1 (N). (O) Frequency of nuclear-localized synergid fluorescence after fertilization: wild-type (n = 328) and ein3 eil1 (n = 320). (P and Q) Cleared whole mounts of wild-type (P) and DD2::EIN3_SRDX (Q). (R) Frequency of four independent DD2::EIN3_SRDX/− transgenic lines containing synergid nuclei after pollination with wild-type. Ovules were scored at the four-nucleate endosperm stage: wild-type (n = 172), EIN3_SRDX/−(1) (n = 108), EIN3_SRDX/−(2) (n = 106), EIN3_SRDX/−(3) (n = 141), EIN3_SRDX/−(4) (n = 142), and ein3 eil1 (n = 106). Black arrowhead points to pollen tube, arrow indicates synergid nucleus, asterisk shows egg cell in (A)–(E) or zygote nucleus in (G)–(Q), and white arrowhead points to central cell in (A)–(E) or endosperm nuclei in (G)–(Q). Scale bars, 25 μm (A, B, D, E, G, H, J, K, M, N, P, and Q) and 10 μm (insets in K and Q). Error bars, mean ± SEM. See also Figure S1. Developmental Cell 2013 25, 310-316DOI: (10.1016/j.devcel.2013.04.001) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 PCD and Fertilization Are Uncoupled in ein3 eil1 Mutants (A) Pollen tube attraction in ein3 eil1 mutants. Wild-type and ein3 eil1 plants were pollinated with pollen of pLAT52::GUS-expressing plants. Ovules were analyzed 24 hr after pollination. (B–G) CLSM images of fertilized wild-type (B–D) and ein3 eil1 ovules (E–G). Two different layers (B and C and E and F, respectively) and the overlay (D and G) are shown. (H) Fertility of different crosses between wild-type and ein3 eil1 plants. (I and J) Expression of a paternally introgressed pAt5g40260::NLS_tdTomato reporter in wild-type (I) and ein3 eil1 seeds (J). (K) Frequencies of wild-type and ein3 eil1 expressing pAt5g40260::NLS_tdTomato in zygote and endosperm (red bar) or zygote only (blue bar). Gray bars indicate seeds without fluorescence signal (wild-type, n = 285; ein3 eil1, n = 282). White arrows point to persistent synergid nucleus. Asterisks indicate zygote nucleus. Black arrowheads show degenerating synergid. White arrowheads point to endosperm nucleus. Scale bars, 25 μm. Error bars, mean ± SEM. See also Figure S2. Developmental Cell 2013 25, 310-316DOI: (10.1016/j.devcel.2013.04.001) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 Premature Ethylene Signaling Can Bypass the Fertilization Requirement for Synergid PCD (A and B) EIN3_YFP localization after pollination. Dark bar indicates EIN3_YFP in synergid, zygote, and endosperm nuclei in the presence of a paternally introgressed tdTomato as shown in (B). Light bar shows EIN3_YFP in synergid nuclei in the absence of tdTomato (n = 156). (C–E) Microinjection of FGR 7.0 ovules. Microinjection needle (red triangle) and ovule prior (C) and after (D) injection. (E) Microinjected ovule (yellow arrow) and noninjected ovule. (F–H) Cleared whole mounts of control- (F) and ACC-injected (G and H) female gametophytes 24 hr after microinjection, exhibiting deformed (G) or no synergid nuclei (H). (I) Frequencies of synergid defects (−ACC, n = 63; +ACC, n = 54). (J and K) Frequency of ctr1 synergid nuclei that differ in size (J) or are fragmented (K) (wild-type, n = 210; ctr1, n = 260). (L–P) CLSM of wild-type (L) and ctr1 female gametophytes (M–P). In ctr1 mutants, fragmented nuclei (M–O) and autofluorescing synergids (P) were observed. (N and O) Different optical sections of a magnification of (M). (Q–S) Frequencies of FGR 8.0 expression patterns in wild-type (light-gray bar, n = 180) and ctr1 (dark-gray bar, n = 216) female gametophytes. (Q) Nuclear localization of NLS_3×GFP in both synergids. (R and S) Defective nuclear localization of NLS_3×GFP in one (R) or both (S) synergids. White arrows point to synergid nucleus (sn). Yellow arrow shows microinjected ovule. Black arrowheads point to degenerating synergid. Asterisks indicate egg cell. White arrowheads show central cell nucleus. Scale bars, 25 μm (B, E–H, L, M, and P–S), 50 μm (C and D), and 12.5 μm (N and O). Error bars, mean ± SEM. See also Figure S3. Developmental Cell 2013 25, 310-316DOI: (10.1016/j.devcel.2013.04.001) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 Reprogramming of Synergid Nuclei in ein3 eil1 Mutants (A and B) ein3 eil1 seeds (B) with two synergid-like nuclei in comparison to wild-type (A). (C) Frequency of ein3 eil1 ovules containing two synergid-like nuclei in the two-nucleate endosperm stage (2 ES) (n = 127) and four-nucleate endosperm stage (4 ES) (n = 157). (D) Frequency of ein3 eil1 seeds expressing the central cell marker pMEA::NLS_tdTomato (red bar, n = 218) and pAtrBohD::NLS_GUS (blue bar, n = 124) in synergid-derived nuclei. (E–H) Wild-type and ein3 eil1 ovules expressing endosperm reporter. (I–P) Dynamics of FGR 7.0 expression in wild-type (I–L) and ein3 eil1 seeds (M–P) 16 hr after manual self-pollination (start of observation = 0 min). Arrows point to synergid or synergid-derived nuclei. Asterisks indicate zygote nucleus. White arrowheads show central cell-derived endosperm nuclei. Scale bars, 25 μm. Error bars, mean ± SEM. See also Movie S1 and Table S1. Developmental Cell 2013 25, 310-316DOI: (10.1016/j.devcel.2013.04.001) Copyright © 2013 Elsevier Inc. Terms and Conditions