An EAR-Dependent Regulatory Module Promotes Male Germ Cell Division and Sperm Fertility in Arabidopsis

Abstract

The production of the sperm cells in angiosperms requires coordination of cell division and cell differentiation. In Arabidopsis thaliana, the germline-specific MYB protein DUO1 integrates these processes, but the regulatory hierarchy in which DUO1 functions is unknown. Here, we identify an essential role for two germline-specific DUO1 target genes, DAZ1 and DAZ2, which encode EAR motif-containing C₂H₂-type zinc finger proteins. We show that DAZ1/DAZ2 are required for germ cell division and for the proper accumulation of mitotic cyclins. Importantly, DAZ1/DAZ2 are sufficient to promote G2- to M-phase transition and germ cell division in the absence of DUO1. DAZ1/DAZ2 are also required for DUO1-dependent cell differentiation and are essential for gamete fusion at fertilization. We demonstrate that the two EAR motifs in DAZ1/DAZ2 mediate their function in the male germline and are required for transcriptional repression and for physical interaction with the corepressor TOPLESS. Our findings uncover an essential module in a regulatory hierarchy that drives mitotic transition in male germ cells and implicates gene repression pathways in sperm cell formation and fertility.

Figure 1.

DAZ1 and DAZ2 Encode Male Germline-Specific EAR Motif–Containing C₂H₂-Type Zinc Finger Proteins. (A) Schematic diagram of the DAZ1 and DAZ2 loci. The locations of three dispersed C₂H₂-type zinc finger domains, a putative nuclear localization signal, a conserved CLLM(^L/_M) motif, and two EAR motifs are marked. T-DNA insertion sites for the daz1-1, daz1-2, and daz2-1 mutant alleles are indicated. (B) Expression of ProDAZ1:DAZ1-mCherry and ProDAZ2:DAZ2-mCherry during pollen development. MSP, microspore; EBC, early bicellular; LBC, late bicellular; TCP, tricellular pollen; MPG, mature pollen grains. Bar = 15 μm. (C) Relative transcript levels of DUO1, DAZ1, and DAZ2 determined by qRT-PCR analysis of microspore (MS), bicellular pollen (BC), tricellular pollen (TC), and mature pollen (MP) samples. Shading indicates the earlier peak of DUO1 transcripts (pink) compared with DAZ1 and DAZ2 (blue). Error bars represent the se of three technical replicates. (D) Developmental expression of DUO1, DAZ1, and DAZ2 fusion proteins in the germline. Fluorescence of developing germ cells from ProDUO1:DUO1-mRFP, ProDAZ1:DAZ1-mCherry, and ProDAZ2:DAZ2-mCherry lines is shown (see Methods). Error bars represent the se. Shading indicates the earlier peak of DUO1 protein (pink) compared with DAZ1 and DAZ2 (blue). EGC, MGC, LGC, early, mid, late germ cell; ESC, MSC, LSC, early, mid, late sperm cell; SC, mature sperm cell.

Figure 2.

MYB Binding Sites Are Essential for DUO1-Dependent Transactivation of the DAZ1 and DAZ2 Promoters. Schematic diagrams (left) of wild-type and mutated DAZ1 (A) and DAZ2 (B) promoter fragments used in trans-activation assays by agroinfiltration of tobacco (Nicotiana tabacum) leaves. The positions of the intact MYB sites remaining are indicated (white boxes). The mean relative activities (FLuc/RLuc) shown are from at least four independent experiments; error bars indicate the se. The “+/−” indicates the presence or absence of 35S:DUO1 expressing strains.

Figure 3.

DAZ1 and DAZ2 Are Essential for Division of the Generative Cell. (A) to (D) Phenotypes of tricellular wild-type (A) and bicellular mutant ([B] to [D]) pollen genotypes stained with DAPI. Bar = 10 μm. (E) to (J) Transmission electron micrographs of wild-type ([E] to [G]) and daz1-1 daz2-1 mutant pollen ([H] to [J]). The boxed regions, magnified left to right, show association of the vegetative cell nucleus and sperm cell (G) or mutant germ cell (J). White arrowheads indicate the nuclear envelope and black arrowheads indicate double membranes enclosing the germline cells. VN, vegetative cell nucleus; GN, mutant germ cell nucleus; SC, sperm cells. Bars = 5 μm. (K) Scatter box plot of the relative DNA content of duo1-4, duo2, and daz1-1 daz2-1 mutant germ cell nuclei normalized to the DNA content (2.0C) of duo2 germ cells (Durbarry et al., 2005). Both duo1-4 and daz1-1 daz2-1 germ cell nuclei had a significantly greater DNA content compared with duo2 (Tukey-Kramer honestly significantly different, P < 0.01) [See online article for color version of this figure.]

Figure 4.

DAZ1/DAZ2 Promotes Germ Cell Division but Not Differentiation in the Absence of DUO1. (A) Wild-type pollen is tricellular with ProHTR10:H2B-GFP expression. (B) duo1-1 pollen is bicellular with no ProHTR10:H2B-GFP activity. (C) Expression of ProDUO1:DAZ1-mCherry in duo1-1 rescues germ cell division, but the HTR10 marker is not expressed. Bar = 5 μm. (D) Frequency of tricellular pollen and ProHTR10:H2B-GFP-expressing (GFP+) pollen in duo1-1 plants, with (+DAZ) or without (-DAZ) the rescuing ProDUO1:DAZ1-mCherry transgene. Error bars represent the se. n ≥ 6 lines. (E) to (G) Condensation of the germ cell nucleus, marked with ProDUO1:H2B-tdTomato, during G2- to M-phase transition. Germ cell chromatin in premitotic nuclei (E) is condensed during prophase (F) and further compacted as chromosomes congress at metaphase (G). Bar = 10 μm. (H) The percentage of mitotic germ cells in developing anthers of wild-type, duo1-4^+/−, daz1-1^−/−, and daz1-1^−/− daz2-1^+/− plants. Error bars represent the se.

Figure 5.

DAZ1 and DAZ2 Are Required for the Accumulation of Mitotic Cyclins. (A) to (C) ProCYCB1;1:MDB-GFP expression during the G2- to M-phase transition. GFP is first detected in nuclei of premitotic cells (A) and increases in cells undergoing mitosis ([B] and [C]). Corresponding nuclear phenotypes are marked with ProDUO1:H2B-tdTomato. Bar = 5 μm. (D) Frequency of premitotic (PM) cells with (PM+, green) and without (PM-, white) ProCYCB1;1:MDB-GFP expression. Anthers enriched with mitotic cells were analyzed from at least two independent lines of wild-type, duo1-4^+/−, daz1-1^−/−, and daz1-1^−/− daz2-1^+/−. Asterisks indicate a significant difference in the ratio observed in duo1-4^+/− anthers compared with the wild type (χ² = 149.77, ***P < 0.001). (E) Reduced accumulation of ProCYCB1;1:MDB-GFP in premitotic germ cells of daz1-1^−/− daz2-1^+/− anthers compared with daz1-1^−/− anthers. Error bars represent the se. n > 25. (F) and (G) Fluorescence (F) and DIC (G) image of mature pollen from duo1-4^+/− plants showing that ProDUO1:MDB-mCherry expression only persists in mutant germ cells. Bar = 30μm. (H) Frequency of germ cells with persistent ProDUO1:MDB-mCherry signal (mCherry+) in mature pollen. Data represent means of T1 lines (n > 6) for duo1-4^+/− and daz1-1^+/− daz2-1^−/−. Error bars represent the se. (I) Frequency of germ cells with a ProDUO1:MDB-mCherry signal in successive bud stages. Anthers were dissected from staged buds of a representative T1 line for duo1-4^+/− and daz1-1^−/− daz2-1^+/−. Gray shading indicates the developmental window of germ cell mitosis. (J) Reduced CYCB1;1 and CYCB1;2 transcript levels in pollen from duo1-4^+/− and daz1-1^+/− daz2-1^−/− plants. Data from qRT-PCR analysis are presented as fold change relative to the wild type, and error bars represent the se of six biological replicates.

Figure 6.

Mutant daz1 daz2 Germ Cells Are Not Competent for Fertilization. (A) and (B) Mature siliques from self fertilized daz1-1^+/− daz2-1^−/− mutant plants (B) contained aborted ovules (white arrowheads) as well as normal seeds similar to those of the wild type (A). Bar = 200 μm. (C) to (F) DIC images of cleared ovules 2 d after pollination with wild-type pollen showing proliferating endosperm (black arrows) and a dermatogen stage embryo ([C] and [D]). Pollination with daz1-1^+/− daz2-1^−/− plants resulted in a class of small ovules with an unfertilized egg and central cell nucleus ([E] and [F]). The black arrowhead indicates a penetrating pollen tube. ECN, egg cell nucleus; CCN, central cell nucleus. Bars = 100 μm. (G) Frequency of ovule phenotypes observed 2 d after pollination in crosses between ms1^−/− pistils and pollen from wild-type, duo1-4^+/−, daz2-1^−/−, and daz1-1^+/− daz2-1^−/− plants. Error bars represent the se (n = 5 siliques). (H) to (J) Time-lapse images showing the discharge of two daz1 sperm cells, followed by syngamy and movement of sperm cell nuclei. Gamete nuclei (red) were labeled with ProDUO1:H2B-tdTomato. Nuclei of female gametophyte accessory cells (green) were labeled with ProACT11:H2B-GFP. Time elapsed shown is 0 min (H), 15 min (I), and 30 min (J). Bar = 20 μm. (K) and (L) Time-lapse images showing the discharge of single daz1 daz2 germ cells and failure of fertilization. The movie was captured with the same experimental setup and originated from the same flower shown in images (H) to (J). Time elapsed shown is 0 min (K), 15 min (L), and 30 min (M). Bar = 20 μm.

Figure 7.

DAZ1/DAZ2-Dependent Pathways Influence Sperm Cell Differentiation. (A) to (F) Images show the expression of gamete differentiation markers in daz1-1 sperm cells (left) and daz1-1 daz2-1 germ cells (right). The levels of fluorescence are shown in the corresponding bar chart. Relative to daz1-1 sperm cells, marker expression was reduced in daz1-1 daz2-1 germ cells for ProHTR10:H2B-GFP ([A] and [B]; Welch’s t test, **P < 0.01) and ProHAP2:HAP2-eYFP ([C] and [D]; Welch’s t test, ****P < 0.0001), and increased for ProDUO1:DUO1-mCherry ([E] and [F]; Welch’s t test, ***P < 0.001). Error bars represent the se, n > 30. Bars = 10 μm. (G) Relative transcript levels of gamete differentiation genes in pollen from duo1-4^+/− and daz1-1^+/− daz2-1 plants determined by qRT-PCR analysis. HTR10, HAP2, TIP5;1, OPT8, and PCR11 are all germline-specific DUO1 target genes, and MYB101 is a vegetative cell-specific gene. Values are shown as fold change difference relative to the wild type. Error bars represent the se of six biological replicates.

Figure 8.

DAZ1 Function Involves EAR-Mediated Mechanisms That Are Important for Male Germline Development. (A) Schematic diagram of the DAZ1 protein and DAZ1 variants in which the tandem EAR motifs (gray shading) are mutated independently (mEAR-1 and mEAR-2) and in combination (mEAR-1,2) or both deleted (ΔEAR-1,2). (B) Yeast two-hybrid analysis of TPL interaction with DAZ1 and DAZ2. The N-terminal region of TPL (amino acids 1 to 226) was used as prey and DAZ1 variants as bait. Values represent the mean β-galactosidase activity of three technical replicates and error bars the sd. (C) DAZ1 repression activity in Arabidopsis protoplasts. The luciferase reporter gene is driven by a chimeric promoter containing multiple GAL4 and ERF5-responsive binding sites (Ohta et al., 2001). Relative luciferase activity after cotransfection with 35S-ERF5 and each 35S-GAL4DB-DAZ1 variant is presented. Values represent the mean of four technical replicates and error bars the se. (D) Functional analysis of the EAR motifs in DAZ1 by in planta complementation. The ProDAZ1:DAZ1-mCherry transgene and mEAR variants were introduced into daz1-1^−/− daz2-1^+/− plants. Rescue of germ cell division was determined from the percentage of tricellular pollen, whereas rescue of male transmission was determined from the inheritance of transgene-linked antibiotic resistance. Results are presented as rescue efficiency relative to full-length DAZ1 protein, and error bars represent the se. n = 4 T1 lines for analysis of division rescue and n > 200 seedlings for analyses of male transmission.

Figure 9.

The DAZ1/DAZ2 Regulatory Node Is Essential for Sperm Cell Development. Expression of DUO1 in generative (germ) cells activates genes required for gamete specification, including DAZ1 and DAZ2. DAZ1/DAZ2 occupy a distinct node in the DUO1 network to counteract repressive mechanisms (R) via EAR-dependent interaction with the corepressor TPL. DAZ1/DAZ2 overcomes a developmental block to sperm cell formation and maturation by (1) facilitating DUO1-dependent activation of gamete differentiation genes and the turnover of DUO1 and (2) the G2- to M-phase accumulation of mitotic cyclins to promote germ cell division.