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First published online 25 July 2007
doi: 10.1242/dev.004788

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Haploinsufficiency after successive loss of signaling reveals a role for ERECTA-family genes in Arabidopsis ovule development

Lynn Jo Pillitteri, Shannon M. Bemis, Elena D. Shpak^* and Keiko U. Torii

Department of Biology, University of Washington, Seattle WA 98195 USA.

View larger version (52K): [in this window] [in a new window]   Fig. 1. Growth phenotype of wild-type and er-family mutant Arabidopsis. (A-H) Wild-type and er-family mutant inflorescence stems. (A) Wild type (WT); (B) er-105; (C) er-105 erl1-2; (D) er-105 erl1-2 erl2-1; (E) er-105 erl1-2 erl2-1/+; (F) er-105 erl1-2/+ erl2-1; (G) er-105 erl1-4 erl2-1/+; and (H) er-105 erl1-1 erl2-1/+. Wild type and all combination mutants (A-C,E-F) except er-105 erl1-2 erl2-1 triple mutant (D) maintain proper floral organ patterning and elongation. Among the combinations, er-105 erl1-2 erl2-1/+ (E), er-105 erl1-4 erl2-1/+ (H) and the triple mutant (D), are female sterile. Scale bar: 1 cm. (I) Schematic of the ERL1 gene with location of T-DNA insertions for erl1-1 and erl2-1, and the point mutation for erl1-4. Exons are indicated as vertical bars, introns as lines. The T-DNA right border (RB) is boxed. (J) RT-PCR analysis of four separate erl1-1 plants with reduced levels of ERL1 transcript compared with wild type. ACTIN 2 serves as a positive control.

View larger version (85K): [in this window] [in a new window]   Fig. 2. Phenotypic effects of loss of er-family function on ovule development. (A-D) Scanning electron micrographs (SEMs) of Arabidopsis ovules at anthesis. (A) Wild type; (B) er-105 erl1-2/+ erl2-1; (C) er-105 erl1-1 erl2-1/+; (D) er-105 erl1-2 erl2-1/+. In er-105 erl1-2 erl2-1/+, both inner and outer integuments are shorter leaving the nucellus exposed (arrows). (E,F) Sagittal sections through the gametophyte of a er-105 erl1-2/+ erl2-1 (E) and er-105 erl1-2 erl2-1/+ (F) ovule at anthesis. In the er-105 erl1-2 erl2-1/+ mutant, the gametophyte has degenerated into a mass of small cells. (G) SEM of a developing er-105 erl1-2 erl2-1/+ stage 2-I ovule. Both the inner and outer integument initiate as smooth rings at the base of the nucellus, with the outer integument initiating asymmetrically as in wild type. (H-K) In situ hybridization for (H,I) PHB and (J,K) WUS expression. No difference in PHB or WUS expression is detected in mutant ovules, indicating normal regional specification. No signal was detected with PHB or WUS sense probe. f, funiculus; es, embryo sac; ii, inner integument; oi, outer integument; n, nucellus. Scale bars: 20 µm.

View larger version (152K): [in this window] [in a new window]   Fig. 3. Developmental series of ovule development in wild-type, er-105 erl1-2/+ erl2-1 and er-105 erl1-2 erl2-1/+ Arabidopsis. (A-L) DIC images of ovules at stages: 1-II (A,E,I); 2-III (B,F,J); 3-I II (C,G,K) and 4-I (D,H,L). Differences in development are seen at stage 3-I-II (C,G,K). Asymmetric growth of the outer integument occurs in all genotypes; however, er-105 erl1-2 erl2-1/+ integuments appear less organized and gametophyte development has ceased (K). By anthesis (D,H,L), the integuments of er-105 erl1-2 erl2-1/+ are short resulting in the nucellus protruding from the integuments (L). By contrast, the er-105 erl1-2/+ erl2-1 ovule has nearly normal appearance, with a very subtle phenotype (H). (M,N) er-105 erl1-2 erl2-1/+ ovule at stage 2-V with tetrad (M, bracket) and stage 3-II (N) showing two-nuclear gametophyte. MMC, megaspore mother cell; n, nucleus; ii, inner integument; oi, outer integument. Scale bars: 20 µm.

View larger version (49K): [in this window] [in a new window]   Fig. 4. ERL2 is haploinsufficient to maintain integument growth. (A,B) Transverse (horizontal) sections of Arabidopsis ovules at anthesis. (A) er-105 erl1-2/+ erl2-1 has a fully encased and developed embryo sac. (B) er-105 erl1-2 erl2-1/+ ovule has shorter integuments, protruding nucellus with no observable embryo sac. (C) Mean cell area (±s.e.m.) of the outer integument cells of wild type and er-105 erl1-2 erl2-1/+ (n=25, Student's t-test, P=0.331). (D) Cell number (±s.e.m.) in epidermis of ovules (stage 1-II) and the outer layer of the outer integument (stage 2-III, 3-III and 4-I) of wild-type and er-family mutant ovules at the indicated stages (n=25). Asterisks indicate a significant difference from the respective wild type within each stage using Student's t-test (P=0.01). At stage 3-III, a decrease in the number of cells in the outer integument is detected between wild type and er-family mutants. By stage 4-II, the number of cells in the outer integument is dramatically reduced compared with wild type. (E) Real-time RT-PCR analysis of selected cyclins in stage 12 carpels (left) and stage 3-II ovules (right). Loss of ER-family function results in a disruption of cyclin gene expression. Scale bars: 20 µm.

View larger version (79K): [in this window] [in a new window]   Fig. 5. The ER family of LRR-RLKs are expressed in ovules throughout development. (A,B) proERL1::GUS expression in wild-type Arabidopsis. (C,D) proERL2::GUS expression in wild type. (E,F) proERL1::GUS expression in er-105. (G,H) proERL2::GUS expression in er-105. ERL1 and ERL2 promoter activity is higher in the er-105 background than in wild type. Scale bar: 20 µm.

View larger version (88K): [in this window] [in a new window]   Fig. 6. ERL1 and ER expression patterns. (A-H) In situ hybridization in wild-type Arabidopsis using an ERL1 (A-D) or ER (E-H) antisense probe. Both show broad expression in ovules with increased signal intensity in the chalazal region (A,E, arrowheads) and developing integuments (B,F, asterisks). Ovule is outlined in B. (D,H) Signal is still visible at maturity. (I-P) Comparison of ERL1 (I,J,M,N) or ER (K,L,O,P) mRNA accumulation in wild type and ER-family mutants examined by in situ hybridization. (Q) Real-time RT-PCR analysis of ER, ERL1 and ERL2 in stage 12 carpels of wild type and ER-family mutants. Transcript abundance of ER, ERL1 and ERL2 are higher in the absence of the other family members. Scale bars: 20 µm.

View larger version (74K): [in this window] [in a new window]   Fig. 7. Genetic interactions of ER-family genes with PFS2. (A-D) SEMs of mature Arabidopsis ovules. (A) pfs2-1 ovules have aberrant integument development. (B) er-105 erl1-2 erl2-1/+ ovules. (C,D) er-105 erl1-2 erl2-1/+ pfs2-1 mutant ovules have variability in integument development with reduced growth of the outer integument and some bifurcation of developing integuments. (E-G) DIC images of mature ovules. (E) pfs2-1 ovules have some partially developed embryo sacs. (F) er-105 erl1-2 erl2-1/+ has a penetrant phenotype of a small mass of cells in place of the embryo sac. (G) er-105 erl1-2 erl2-1/+ pfs2-1 mutant ovules display variability in embryo sac development similar to the pfs2-1 single mutant. Embryo sac nuclei are indicated by white arrowheads. (H-K) In situ hybridization in the pfs2-1 mutant using ERL1 (H,I) and ER (J,K) antisense probes. (L,M) In situ hybridization in wild type using a PFS2 antisense probe. (N,O) In situ hybridization in er-105 erl1-2 erl2-1/+ using a PFS2 antisense probe. (P,Q) Extended exposure of PFS2 antisense probe in wild type and er-105 erl1-2 erl2-1/+. Intense signal was observed in the inner integuments (arrowheads). No signal was detected with sense probes for ERL1, ER or PFS2. Scale bars: 20 µm. (R) Real-time RT-PCR analysis of PFS2 expression in stage 3-II ovules, given as fold-difference relative to wild type. *, Raw data collected as PFS2 transcript abundance relative to ACTIN 2 (wild type versus er-105 erl1-2 erl2-1/+. Experiment 1: 0.205±0.04 versus 0.351±0.02; Student's t-test P=0.03; 1.72 fold increase in er-105 erl1-2 erl2-1/+. Experiment 2: 0.141±0.003 versus 0.603±0.04; Student's t-test P=0.001; 4.2 fold increase in er-105 erl1-2 erl2-1/+).