The ubiquitin-specific protease subfamily UBP3/UBP4 is essential for pollen development and transmission in Arabidopsis

Abstract

Deubiquitinating enzymes are essential to the ubiquitin (Ub)/26S proteasome system where they release Ub monomers from the primary translation products of poly-Ub and Ub extension genes, recycle Ubs from polyubiquitinated proteins, and reverse the effects of ubiquitination by releasing bound Ubs from individual targets. The Ub-specific proteases (UBPs) are one large family of deubiquitinating enzymes that bear signature cysteine and histidine motifs. Here, we genetically characterize a UBP subfamily in Arabidopsis (Arabidopsis thaliana) encoded by paralogous UBP3 and UBP4 genes. Whereas homozygous ubp3 and ubp4 single mutants do not display obvious phenotypic abnormalities, double-homozygous mutant individuals could not be created due to a defect in pollen development and/or transmission. This pollen defect was rescued with a transgene encoding wild-type UBP3 or UBP4, but not with a transgene encoding an active-site mutant of UBP3, indicating that deubiquitination activity of UBP3/UBP4 is required. Nuclear DNA staining revealed that ubp3 ubp4 pollen often fail to undergo mitosis II, which generates the two sperm cells needed for double fertilization. Substantial changes in vacuolar morphology were also evident in mutant grains at the time of pollen dehiscence, suggesting defects in vacuole and endomembrane organization. Even though some ubp3 ubp4 pollen could germinate in vitro, they failed to fertilize wild-type ovules even in the absence of competing wild-type pollen. These studies provide additional evidence that the Ub/26S proteasome system is important for male gametogenesis in plants and suggest that deubiquitination of one or more targets by UBP3/UBP4 is critical for the development of functional pollen.

Figure 1.

Gene structure and genetic analysis of Arabidopsis UBP3 and UBP4. A, Gene structures showing the positions of the coding regions for the Cys, Gln, Gly, Leu, Phe, and His boxes, the positions of the ubp3-1 and ubp4-1 T-DNA insertions, and primer binding sites used for genotyping and RT-PCR analyses. Lines represent introns and nontranscribed DNA upstream and downstream of each gene. White and gray rectangles represent protein coding regions and 5′- and 3′-untranslated regions, respectively. The nucleotide sequence of each primer is presented in “Materials and Methods.” B, Bar graph depicting microarray expression data of UBP3 (black bars) and UBP4 (gray bars) as reported by Honys and Twell (2004). UNM, Uninucleate microspores; BCP, bicellular pollen; TCP, tricellular pollen; and MPG, mature pollen grains. C, RT-PCR analysis of total RNA from wild-type Ws plants and plants homozygous for either the ubp3-1 or ubp4-1 T-DNA insertions. Genomic DNA from wild-type Ws plants confirmed the activity of the primers, whereas RT-PCR analysis of the UBP14 transcripts verified the integrity of the initial RT reactions. D, Phenotype of 3-week-old ubp3-1 and ubp4-1 plant seedlings as compared to wild-type Ws Arabidopsis.

Figure 2.

ubp3-1 ubp4-1 double-mutant pollen is defective in pollen transmission. A, Punnett square diagrams of the reciprocal crosses. Upper and lower cases represent the wild-type and mutant alleles, respectively. Possible pollen genotypes are indicated to the left of the squares, possible ovule genotypes above the squares, and possible offspring genotypes within the squares. The gray X in the progeny square marks the UBP3/ubp3-1; UBP4/ubp4-1 genotype that was not found when pollen from a UBP3/ubp3-1; ubp4-1/ubp4-1 donor was used. B and C, PCR genotyping of 20 individual offspring resulting from each reciprocal cross. DNA isolated from individual offspring was genotyped at the UBP3 locus using two PCR reactions: UBP3-2 + UBP3-4 to detect the presence of a wild-type allele (UBP3) and UBP3-2 + JL-202 to detect the presence of a mutant ubp3-1 allele (ubp3-1). DNA from homozygous mutant ubp3-1 (m) or wild-type (w) individuals was used as control. See Figure 1 for the positions of the primers.

Figure 3.

The mutant UBP3-C32S-T transgene is transcribed. Total RNA isolated from wild-type Ws, homozygous ubp3-1/ubp3-1; UBP4/UBP4 plants, and ubp3-1/ubp3-1; UBP4/ubp4-1 plants containing the UBP3-C32S-T transgene were subjected to RT-PCR analysis using primers 3-5 and 3-6. + and −, RT reaction was carried out with or without reverse transcriptase, respectively. Lane gDNA indicates the PCR product using genomic DNA as a template and lane M contains a DNA size ladder with approximate lengths indicated on the right. The arrow designates the RT-PCR-amplified UBP3 cDNA product.

Figure 4.

Germination and nuclei content of ubp3-1 ubp4-1 pollen. A, In vitro germination of pollen tetrads from UBP3/ubp3-1; ubp4-1/ubp4-1; qrt1-2/qrt1-2 plants. Each image shows an individual tetrad where three or four pollen grains germinated. The bar represents 50 μm. B to G, DAPI staining of pollen from UBP3/UBP3; ubp4-1/ubp4-1; qrt1-2/qrt1-2 (33) and UBP3/ubp3-1; ubp4-1/ubp4-1; qrt1-2/qrt1-2 (33) plants showing that some pollen grains contain two sperm nuclei, others only one generative/sperm nucleus, and some without any visible sperm nuclei (left). Photographs of each pollen tetrad under visible light (right) are shown. Fluorescence of the DAPI under UV light. B, Tetrad from a UBP3/UBP3; ubp4-1/ubp4-1; qrt1-2/qrt1-2 plant where each pollen grain contains two sperm nuclei. Shown are tetrads from a UBP3/ubp3-1; ubp4-1/ubp4-1; qrt1-2/qrt1-2 plant where four (C), three (D), or two (E and F) pollen grains contain two sperm nuclei, but the remaining pollen grains most often contain only one generative/sperm nucleus. G, Tetrad from a UBP3/ubp3-1; ubp4-1/ubp4-1; qrt1-2/qrt1-2 plant where one pollen grain contains two sperm nuclei, one pollen grain contains only a vegetative nucleus, and the other two pollen grains are visibly shrunken and do not show any nuclei.

Figure 5.

Transmission electron microscope analysis of high-pressure frozen/freeze-substituted pollen. The endomembrane organization of pollen from qrt1-2/qrt1-2 (A), ubp3-1/ubp3-1; UBP4/ubp4-1; qrt1-2/qrt1-2 (D), and UBP3/ubp3-1; ubp4-1/ubp4-1; qrt1-2/qrt1-2 (G) plants appears similar following pollen mitosis I. Upper case and lower case numbers indicate wild-type and mutant alleles, respectively. D, Pollen tetrad containing two pollen grains in which the generative cell (GC) is still attached to the intine wall. The pollen grain depicted in G is slightly more mature in that the generative cell has already been internalized. VN, Vegetative nucleus. Pollen from qrt1-2/qrt1-2 (B and C), ubp3-1/ubp3-1; UBP4/ubp4-1; qrt1-2/qrt1-2 (E and F), and UBP3/ubp3-1; ubp4-1/ubp4-1; qrt1-2/qrt1-2 plants (H–K) following anther dehiscence show differences in vacuole and endomembrane organization. B and C, Pollen from qrt1-2/qrt1-2 plants contain dense cytoplasm packed with lipid bodies (LB), vesicles (V), mitochondria (M), and endoplasmic reticulum (ER) membranes. E, F, H, and I, Pollen grains from ubp3-1/ubp3-1; UBP4/ubp4-1; qrt1-2/qrt1-2 (E and F) and UBP3/ubp3-1; ubp4-1/ubp4-1; qrt1-2/qrt1-2 (H and I) plants showing an enlarged vacuolar system (asterisks). J and K, Collapsed pollen grains from UBP3/ubp3-1; ubp4-1/ubp4-1; qrt1-2/qrt1-2 plants. Scale bars in A, B, D, E, G, H, J, and K = 5 μm; in C, F, and I = 2 μm.