UBIQUITIN-SPECIFIC PROTEASE 26 is required for seed development and the repression of PHERES1 in Arabidopsis

Abstract

The Arabidopsis mutant Atubp26 initiates autonomous endosperm at a frequency of approximately 1% in the absence of fertilization and develops arrested seeds at a frequency of approximately 65% when self-pollinated. These phenotypes are similar to those of the FERTILIZATION INDEPENDENT SEED (FIS) class mutants, mea, fis2, fie, and Atmsi1, which also show development of the central cell into endosperm in the absence of fertilization and arrest of the embryo following fertilization. Atubp26 results from a T-DNA insertion in the UBIQUITIN-SPECIFIC PROTEASE gene AtUBP26, which catalyzes deubiquitination of histone H2B and is required for heterochromatin silencing. The paternal copy of AtUBP26 is able to complement the loss of function of the maternal copy in postfertilization seed development. This contrasts to the fis class mutants where the paternal FIS copy does not rescue aborted seeds. As in the fis class mutants, the Polycomb group (PcG) complex target gene PHERES1 (PHE1) is expressed at higher levels in Atubp26 ovules than in wild type; there is a lower level of H3K27me3 at the PHE1 locus. The phenotypes suggest that AtUBP26 is required for normal seed development and the repression of PHE1.

Figure 1.—

Phenotypes of Atubp26. (A) Col wild type with fertile flowers and elongated siliques. Inset is an anther with pollen grains. (B) Siliques on an early-emerged branch (left) are longer than that of a late-emerged branch (right) in Atubp26-2 due to the lack of dehiscent anthers. Right inset shows a nondehiscent anther from an open flower in a late-emerging branch. Left inset shows a dehiscent anther from a Atubp26-2 homozygote from an open flower in an early-emerging branch. (C) Col mature seeds. (D) Atubp26-2 homozygote seeds showing both full and shriveled seeds. (E) Atubp26-3 homozygote seeds. (F) Atubp26-4 homozygote seeds. (G) An unfertilized ovule 7 days postemasculation in Col showing a fused central-cell nucleus. (H) An Atubp26-2 unfertilized ovule with six endosperm nuclei (two out of focus). (I) An Atubp26-3 unfertilized ovule with four endosperm nuclei. (J) An Atubp26-4 unfertilized ovule with endosperm nuclei (some out of focus). (K) An unfertilized ovule with endosperm nuclei of an Atubp26-2 heterozygote in the ap3/ap3 background. (L) An unfertilized ovule with endosperm nuclei of an Atubp26-2 heterozygote in the ap3/ap3 background. Note that the nuclei are concentrated at the micropylar region. (M) An Atubp26-2 aborted ovule with no developed embryo sac. (N) An Atubp26-2 homozygote pollinated with wild-type pollen showing viable seeds. Arrows in H–L indicate the endosperm nuclei. Bars in A and B, 10 mm; C–F and N, 0.5 mm; G–M, 0.1 mm.

Figure 2.—

Mapping and expression of AtUBP26. (A) The mutant gene was mapped to a contig containing At3g49600/AtUBP26. Markers CIW4 and T16K5 FR were used to identify five and one recombinants, respectively, which showed crossovers between the mutant locus and the markers. Markers T32N15 FR, T21J18 FR, and F2K15 FR identified seven, one, and one recombinants, respectively. (B) Schematic of AtUBP26 showing the T-DNA insertions in three alleles. Solid boxes represent exons and open boxes, introns. (C) RT–PCR showing that AtUBP26 is expressed in buds, cauline leaves (CL), rosette leaves (RL), young seedlings (YSe), seeds 3 days postfertilization (Se3), embryos 7 days postfertilization (Em7), endosperm 7 days postfertilization (En7), young siliques 1–2 days postfertilization (YSi1-2), and stems.

Figure 3.—

Atubp26 aborted seed phenotypes. (A–C) Embryo and endosperm development in wild-type seeds at 48, 72, and 96 hr postfertilization. In A and B, globular embryos and syncytial endosperm were observed. In C, heart-stage embryo and cellularized endosperm were observed. (Inset) Cell wall of endosperm. (D and E) Atubp26 seeds at 48 and 72 hr showing similar development to wild type of the same age. (F) An Atubp26 seed at 96 hr containing an arrested globular embryo and syncytial endosperm. (Inset) Free endosperm nuclei. (G and H) Atubp26 seeds at 120 hr containing arrested heart or globular embryo and syncytial endosperm. (I) An Atubp26 seed at 120 hr containing a torpedo stage embryo. (J) Seed with arrested globular embryo from a heterozygote. (K) Seed with arrested heart embryo from a heterozygote. Arrows indicate the embryos. Bars in A, B, D, and E are 50 μm; in C, F, and G–I are 0.1 mm.

Figure 4.—

PHE1 expression is elevated in Atubp26 siliques. (A) Real-time PCR showing PHE1 expression in Atubp26 and wild-type siliques at 36, 60, and 84 hr postfertilization. (B) Chromatin immunoprecipitation analysis of H3K27me3 at the PHE1 locus. There was a higher enrichment of the PHE1 region in Col than in Atubp26 siliques (60–84 hr old) after chromatin immunoprecipitation using a H3K27me3 antibody but no difference in buds. (C) MEA, FIS2, FIE, AtMSI1, and FWA expression in Atubp26 and wild-type siliques of 2–3 days; PHE1 expression in Atubp26 and wild-type leaves; PHE1 expression in mea and wild-type siliques of 2–3 days.