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. 2012;7(1):e29470.
doi: 10.1371/journal.pone.0029470. Epub 2012 Jan 9.

Mutation in SUMO E3 ligase, SIZ1, disrupts the mature female gametophyte in Arabidopsis

Yu Ling  1 , Chunyu Zhang, Tong Chen, Huaiqing Hao, Peng Liu, Ray A Bressan, Paul M Hasegawa, Jing Bo Jin, Jinxing Lin
Affiliations

Mutation in SUMO E3 ligase, SIZ1, disrupts the mature female gametophyte in Arabidopsis

Yu Ling et al. PLoS One. 2012.

Abstract

Female gametophyte is the multicellular haploid structure that can produce embryo and endosperm after fertilization, which has become an attractive model system for investigating molecular mechanisms in nuclei migration, cell specification, cell-to-cell communication and many other processes. Previous reports found that the small ubiquitin-like modifier (SUMO) E3 ligase, SIZ1, participated in many processes depending on particular target substrates and suppression of salicylic acid (SA) accumulation. Here, we report that SIZ1 mediates the reproductive process. SIZ1 showed enhanced expression in female organs, but was not detected in the anther or pollen. A defect in the siz1-2 maternal source resulted in reduced seed-set regardless of high SA concentration within the plant. Moreover, aniline blue staining and scanning electron microscopy revealed that funicular and micropylar pollen tube guidance was arrested in siz1-2 plants. Some of the embryo sacs of ovules in siz1-2 were also disrupted quickly after stage FG7. There was no significant affects of the siz1-2 mutation on expression of genes involved in female gametophyte development- or pollen tube guidance in ovaries. Together, our results suggest that SIZ1 sustains the stability and normal function of the mature female gametophyte which is necessary for pollen tube guidance.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Expression of the Prosiz1::GUS–GFP gene.
(A) Expression of Prosiz1::GUS–GFP in the whole inflorescence. A GUS signal was detected in most of the flowers, except the latest ones, while the strongest GUS signal was found in sepals. The inflorescence in (A) was stained with 1 mM 5-bromo-4-chloro-3-indolyl-b-glucuronic acid (X-Gluc) for 12 h. (B) GUS signal in a flower after pollination. The style of the pistil was stained strongly by X-Gluc, and the upper part of the carpel and the stem of the stamen were also stained by X-Gluc. No GUS signal was seen in the anthers. (C) GUS signal in reproductive organs at the end of pollination. The style was strongly stained by X-Gluc, while no GUS signal was seen in the stigmatic cells or pollen. (D) No GUS signal was detected in the whole flower in the wild-type plants after staining with 1 mM X-Gluc for 12 h. (E) Expression of Prosiz1::GUS–GFP could be detected in all cells within the ovule before fertilization after staining with 1 mM X-Gluc for 8 h. (F) GFP fluorescence of Prosiz1::GUS–GFP can be seen in all cells of the ovules. (G) Wild-type ovule control. No fluorescence was detected in the wild-type ovule under LSCM. Pg, pollen grain; Sc, stigmatic cell; St, style.
Figure 2
Figure 2. Silique development and seed-set of siz1-2, nahG siz1-2, wild-type, and the Prosiz1::SIZ1-GFP construct-transformed siz1-2 mutant plants (SSG).
(A) Siliques of siz1-2, nahG siz1-2, wild-type, and SSG 8–10 days after pollination. (B) Dissected silique from siz1-2 homozygous plants showing severely reduced seed-set and undeveloped ovules. Similar results were also found in line siz1-3 (data not shown). De, defective embryo. (C) Dissected silique from nahG siz1-2 plants showing severely reduced seed-set and undeveloped ovules, similar to siz1-2. De, defective embryo. (D) Dissected silique of a wild-type plant with a full seed-set. (E) Dissected silique of a SSG plant with full seed-set, similar to that of the wild-type plant. (F) Percentage of defective embryos in siz1-2, nahG siz1-2, Col-0, and SSG pistils. A mean value of three repeats, asterisks indicate no significant difference between percentage of defective embryos of siz1-2 and nahG siz1-2 (P<0.05).
Figure 3
Figure 3. Analysis of ovule development and in vitro germination of siz1-2 pollen grains compared to the wild type by DIC microscopy.
(A)–(C) Ovules in a siz1-2 mutant under a DIC microscope. The fertilized ovule grew bigger and formed a quadrant embryo (Em) within the embryo sac (B); the unfertilized ovule stopped growing, with no proembryo appearing. (D) Wild-type pollen tubes cultured at 28°C in vitro. Pollen tubes with normal morphology are indicated by an arrow. (E) siz1-2 pollen tubes incubated under the same condition as (A), showing no obvious difference compared to the wild-type pollen tube. Em, embryo. Pg, pollen grain. Pt, pollen tube. Bar = 50 µm in (A), 8 µm in (B) and (C), 200 µm in (D) and (E).
Figure 4
Figure 4. Pollen tube growth in the pistils from wild-type and homozygous siz1-2 plants.
(A)–(G) decolorized aniline blue staining of pistils 1–2 days after pollination (DAP). (A) The whole scene of pollen tube growth within the siz1-2 pistil. (B) The whole scene of pollen tube growth within the wild-type pistil. (C) The fertilized wild-type ovules showing pollen tubes (arrow) grew into the micropyle (arrowhead) and became bigger in volume. The pistil was harvested 2 days after pollination. (D) Mutant ovules without a pollen tube growing toward the funiculus, while many pollen tubes grew within the placenta. (E) The mutant ovule with pollen tube (arrow) growing around the funiculus, but turning away from the funiculus, without targeting the micropyle (arrowhead). (F) Two undeveloped ovules with pollen tubes (arrow) growing around the funiculus without targeting the micropyle. (G) Representative mutant ovule with pollen tube growing near the micropyle opening but failing to target the female gametophyte. (H)–(M) Scanning electron microscopy analysis of pistils 1–2 days after pollination. (H) Scanning electron micrograph of wild-type ovules showing that pollen tubes grew along the funiculus and then entered the micropyle (arrowhead). (I) Scanning electron micrograph of some siz1-2 ovules showing that pollen tubes grew along the funiculus and then entered the micropyle (arrowhead), similar to those of the wild type. (J)–(M) Aberrant pollen tube guidance in siz1-2 ovules. (L) A pollen tube stopped growing near the micropyle (arrowhead). (J) A pollen tube bypassing the micropyle and growing on the surface of the integument. (K) A pollen tube grew along the funiculus but failed to enter the micropyle and turned away. (M) An example showing that no pollen tube grew on the funiculus of the ovule. St, style; Tt, pollen tube transmitting tract. Arrows indicate pollen tubes and arrowheads show micropyle. Bar = 200 µm in (A) and (B), 40 µm in (C)–(M).
Figure 5
Figure 5. Ovule development from stage FG1 to FG7 in the wild type and siz1-2 mutant.
The upper panels show ovule development of the wild type, as revealed by laser scanning confocal microscope, while the lower panels show ovule development of the siz1-2 mutant. Corresponding development stages of the ovules examined are indicated below. Bar = 20 µm.
Figure 6
Figure 6. Final phenotypes of the female gametophyte in the wild type and siz1-2 mutant.
(A) LSCM images of an ovule derived from a wild-type flower; the pistil was harvested 2 day after emasculation. (B)–(D) LSCM images for ovules derived from siz-1-2 flowers; the pistils were harvested 2 days after emasculation. Percentages of abnormal female gametophytes among the examined ovules are indicated below. Cn, central cell nucleus; En, egg cell nucleus; Sn, synergid cell nucleus. Bar = 40 µm.
Figure 7
Figure 7. Expression patterns of selected genes from siz1-2 and wild-type siliques.
The tubulin α-2 gene (AT1G04820) was used as the internal control, and its expression level was set arbitrarily as 1.
See this image and copyright information in PMC

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