Exportin1 genes are essential for development and function of the gametophytes in Arabidopsis thaliana

Abstract

Gametes are produced in plants through mitotic divisions in the haploid gametophytes. We investigated the role of EXPORTIN1 (XPO1) genes during the development of both female and male gametophytes of Arabidopsis. Exportins exclude target proteins from the nucleus and are also part of a complex recruited at the kinetochores during mitosis. Here we show that double mutants in Arabidopsis XPO1A and XPO1B are gametophytic defective. In homozygous-heterozygous plants, 50% of the ovules were arrested at different stages according to the parental genotype. Double-mutant female gametophytes of xpo1a-3/+; xpo1b-1/xpo1b-1 plants failed to undergo all the mitotic divisions or failed to complete embryo sac maturation. Double-mutant female gametophytes of xpo1a-3/xpo1a-3; xpo1b-1/+ plants had normal mitotic divisions and fertilization occurred; in most of these embryo sacs the endosperm started to divide but an embryo failed to develop. Distortions in male transmission correlated with the occurrence of smaller pollen grains, poor pollen germination, and shorter pollen tubes. Our results show that mitotic divisions are possible without XPO1 during the haploid phase, but that XPO1 is crucial for the maternal-to-embryonic transition.

Figure 1.—

Structure of XPO1 genes, mutant alleles, and expression analysis. (A) Solid boxes, exons; triangles, T-DNA insertions in xpo1a-1 (intron 4, 1304 bp from the transcription initiation start), in xpo1a-2 (intron 9, 2417 bp from the transcription initiation start), in xpo1a-3 (exon 24, 5919 bp from the transcription initiation start), and in xpo1b-1 (exon 1, 74 bp from the transcription initiation start). (B) RT–PCR analysis of xpo1a-1, xpo1a-2, xpo1a-3, xpo1b-1, and wt (wild type) seedling RNA. g, genomic DNA control.

Figure 2.—

Expression analysis of XPO1. RT–PCR analysis of XPO1A and XPO1B expression in p, pollen; u, unpollinated pistils; and s, seedling. ACT2 was used as the control.

Figure 3.—

Phenotype of xpo1 mutant pollen grains. Transmitted light image of pollen grains from a3/+; b1/b1 (A) and a3/a3; b1/+ (C) plants. DAPI-stained pollen grains from a3/+; b1/b1 (B) and a3/a3; b1/+ (D) plants. (E) Size distributions of pollen in wild type (WT) and the a3/+; b1/b1 and a3/a3; b1/+ plants. (F) Percentage of germination, ±SE (n = 3) of pollen grains from wild type, a3/a3, b1/b1, a3/+; b1/b1, and a3/a3; b1/+. (G–I) Germination defect in xpo1 mutant pollen grains. Representative images of germination assays with pollen from wild-type Col-0 (G), a3/+; b1/b1 (H), and a3/a3; b1/+ (I) plants. Bars, 30 μm (A–D) or 100 μm (G–I).

Figure 4.—

Phenotype of xpo1 mutant female gametophytes. (A–D) a3/+; b1/b1. (E–G, I, and J) a3/a3; b1/+. Open mature siliques; arrowheads indicate aborted or undeveloped ovules. (B and F) DAB-stained pistils 3 days after pollination. Genotypes are given for the female gametophyte. (C, D, G, I, and J) DIC images of whole-mounted cleared a3/a3; b1/b1 ovules. Percentages represent the occurrence of the depicted phenotype. Arrowheads show endosperm nuclei in G and I or the zygote in J. (H) Single-ovule PCR to detect the paternal XPO1A allele, 3 days after pollination of a3/a3; b1/+ pistils with wild-type (WT) pollen. Bars, 20 μm except in A and E (500 μm) and in B and F (100 μm).

Figure 5.—

Phenotype of xpo1 mutant female gametophytes. Representative DIC images of a3 b1 embryo sacs from a doubly heterozygous a3/+; b1/+ plant (A), unfertilized embryo sac (B), zygote only (C), and endosperm only (D) arrested zygote with endosperm development. Arrowheads show the zygote. Bars, 40 μm.