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Journal List > Plant Physiol > v.137(1); Jan 2005

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Plant Physiol. 2005 January; 137(1): 297–307.
doi: 10.1104/pp.104.053165.
PMCID: PMC548860
Copyright © 2005, American Society of Plant Biologists
Male Germ Line Development in Arabidopsis. duo pollen Mutants Reveal Gametophytic Regulators of Generative Cell Cycle Progression1[w]
Anjusha Durbarry, Igor Vizir, and David Twell*
Department of Biology, University of Leicester, Leicester LE1 7RH, United Kingdom
*Corresponding author; e-mail twe/at/le.ac.uk; fax 44–(0)–116–252–2791.
Received September 9, 2004; Revised November 13, 2004; Accepted November 15, 2004.
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Abstract
Male germ line development in flowering plants is initiated with the formation of the generative cell that is the progenitor of the two sperm cells. While structural features of the generative cell are well documented, genetic programs required for generative cell cycle progression are unknown. We describe two novel Arabidopsis (Arabidopsis thaliana) mutants, duo pollen1 (duo1) and duo pollen2 (duo2), in which generative cell division is blocked, resulting in the formation of bicellular pollen grains at anthesis. duo1 and duo2 map to different chromosomes and act gametophytically in a male-specific manner. Both duo mutants progress normally through the first haploid division at pollen mitosis I (PMI) but fail at distinct stages of the generative cell cycle. Mutant generative cells in duo1 pollen fail to enter mitosis at G2-M transition, whereas mutant generative cells in duo2 enter PMII but arrest at prometaphase. In wild-type plants, generative and sperm nuclei enter S phase soon after inception, implying that male gametic cells follow a simple S to M cycle. Mutant generative nuclei in duo1 complete DNA synthesis but bypass PMII and enter an endocycle during pollen maturation. However, mutant generative nuclei in duo2 arrest in prometaphase of PMII with a 2C DNA content. Our results identify two essential gametophytic loci required for progression through different phases of the generative cell cycle, providing the first evidence to our knowledge for genetic regulators of male germ line development in flowering plants.
 
Plant sexual reproduction depends on the timely construction of male and female gametes that are produced by the haploid gametophyte generation. In flowering plants, male gametogenesis is restricted to a simple cell lineage of two cell divisions following meiosis that results in the production of two nonmotile sperm cells. The first division of the microspore at pollen mitosis I (PMI) is asymmetric and gives rise to a large transcriptionally active vegetative cell and a diminutive generative cell with condensed chromatin and fewer organelles. After PMI, the two cells follow different developmental pathways that are characterized by the differential control of the cell cycle and gene expression. Whereas the vegetative cell exits the cell cycle in G1 and differentiates, the generative cell completes a further cell cycle to form the two sperm cells (for review, see Tanaka, 1997; Twell et al., 1998). Although the general pathway leading to sperm cell formation is clear, our knowledge of the genetic and molecular control of generative cell cycle progression and male germ line development is very limited.
Gene expression within the male gametes has been explored in some plants. Male gamete specific histones have been identified in isolated generative cells of lily (Lilium longiflorum; Ueda and Tanaka, 1995), and some genes (ERCC1, LGC1, and FtsZ) that are expressed preferentially or specifically in the male gametes have been isolated (Xu et al., 1998, 1999; Mori and Tanaka, 2000). Recently, large scale sequencing of sperm cell cDNAs from maize (Zea mays) has revealed a diverse complement of mRNAs (Engel et al., 2003). Moreover, the expression of cyclin-dependent kinase (CDK), cyclin A1, and histone H3 genes was detected in isolated maize sperm cells (Sauter et al., 1998). Taken together, these data suggest that molecular events related to cell cycle progression are expressed in male gametic cells.
There are two patterns of sperm formation with respect to pollen shed. Sperm cell formation occurs either within the pollen grain or in the pollen tube, and this heterochronic shift is believed to be the outcome of adaptive evolution in angiosperms. A study of almost 2,000 species supports the view that phylogenetically advanced species bearing tricellular pollen have arisen repeatedly from those with pleisomorphic bicellular pollen (Brewbaker, 1967). More detailed studies revealed five patterns of sperm cell development among higher plants that differ with respect to the relative timing of sperm cell formation and further progression of the sperm cell cycle (Friedman, 1999).
Despite the wide range of studies on cell cycle regulation in plants (for review, see Stals and Inze, 2001; Criqui and Genschik, 2002; Dewitte and Murray, 2003), it remains a challenge to identify genes involved in the control of cell cycle progression in specific cell lineages. The simple cell lineage and haploid nature of the male gametophytes provides an important and tractable system in which to identify important cell cycle regulators through mutational analysis. Gametophytic mutants affecting various aspects of pollen development and function in Arabidopsis (Arabidopsis thaliana) have been identified through genetic screens for segregation distortion (Bonhomme et al., 1998; Howden et al., 1998; Grini et al., 1999; Lalanne et al., 2004a, 2004b). Direct screens for morphological pollen mutants have led to the identification of a novel collection of Arabidopsis gametophytic mutants that show cell division defects at PMI (Chen and McCormick, 1996; Twell et al., 2002) or pollen morphogenesis defects (Johnson and McCormick, 2001; Lalanne and Twell, 2002). Here, we describe a new class of gametophytic mutants (duo pollen) that specifically block division of the generative cell during pollen development. We characterize male germ line development and cell cycle progression in duo pollen1 (duo1) and duo pollen2 (duo2) mutants, which map to different locations and affect different phases of the generative cell cycle, providing compelling evidence for male germ line-specific control of cell division.
RESULTS
Genetic Analysis of Two Gametophytic duo pollen Mutations
Pollen released from open flowers of 10,000 M2 plants within an ethyl methanesulfonate mutagenized population was screened for aberrant pollen cell division phenotypes by 4′,6-diamidino-phenylindole (DAPI) staining as described (Park et al., 1998). This screen yielded 6 mutants that produced 35% to 50% pollen grains with only 2 nuclei: a diffuse vegetative nucleus and a more compact generative-like nucleus (Fig. 1Figure 1.). Two of these mutants, duo1 and duo2, that showed highly penetrant pollen phenotypes and no obvious sporophytic phenotypes were selected for detailed genetic and cytological analysis. Both mutants were backcrossed as the female parent to wild-type plants, and approximately 50% of the progeny showed the mutant duo phenotype (Table I). The mutants were isolated as heterozygotes and predicted to act gametophytically. Heterozygous plants harboring a fully penetrant gametophytic mutation produce an equal number of wild-type and aberrant pollen grains. In both duo mutants, approximately one-half of the pollen population were aberrant (Fig. 1, A and BFigure 1.). Tetrad analysis was performed using the qrt1 mutant (Preuss et al., 1994). In qrt1/qrt1 mutants, 100% of tetrads contain 4 pollen grains each with 2 sperm cell nuclei. However, in qrt1 mutants that were also heterozygous for duo1 or duo2 (+/duo;qrt1/qrt1), about 98% of the tetrads contained 2 aberrant members (Fig. 1CFigure 1.) and the remainder 1 aberrant member. The 2:2 segregation of tetrads confirmed that both duo mutations act gametophytically.
Figure 1.
Figure 1.
Figure 1.
Pollen phenotypes of wild-type, duo1, and duo2 mutants. A and B, Light and fluorescence images of DAPI-stained pollen from heterozygous duo1 plants (white arrow heads indicate wild-type and light gray arrow heads mutant pollen). C, Tetrad from qrt1/qrt1 (more ...)
Table I.
Table I.
Genetic transmission of duo mutations
The normal vegetative development, aberrant pollen phenotype, lack of male transmission, and normal female transmission defined both duo mutations as male specific, indicating that these mutations are required specifically for male gametophyte development (Table I). Consistent with the lack of male transmission, screening of backcross populations for both mutants failed to identify duo homozygotes (n > 200). Moreover, examination of mature ovules from heterozygous duo mutants that were cleared and viewed under Nomarski optics did not reveal any embryo sac defects (data not shown).
Tetraploid analysis, in which diploid gametophytes may carry both wild-type and mutant alleles, can be used to determine whether gametophytic mutations are gain- or loss-of-function (Grossniklaus et al., 1998). We induced chromosome doubling in duo mutants by colchicine treatment of diploid heterozygous mutant seed. Tetraploid sectors carrying 2 mutant alleles (duplex) would produce diploid pollen grains either with none, 1, or 2 duo alleles with 1:4:1 gametic ratios, respectively, based on chromosomal segregation alone. However, deviations from a simple 5:1 (no double reduction) ratio of wild-type:mutant pollen to 3.6:1 can be expected based on experimental values of the rate of double reduction (α) in tetraploids that range from 0.0 to 0.3 (Butruille and Boiteux, 2000). Our data showed a 4.1:1 ratio for duo1 and 4.7:1 for duo2, consistent with a duplex recessive model with low rates of double reduction for the corresponding regions of chromosome 3 and 5 (Table II). These results suggest both duo mutations are loss-of-function mutations and that the wild-type functions of DUO1 and DUO2 act to promote generative cell division.
Table II.
Table II.
Tetraploid analysis of duo1 and duo2
duo1 and duo2 were mapped in F2 populations to different chromosomal locations using molecular markers polymorphic between Nossen (No-0) and Columbia (Col-0). duo1 was mapped to chromosome 3 between simple sequence length polymorphism (SSLP) markers RPF24 (79.7 cM) and nga112 (87.9 cM) at position 80.9 ± 0.9 cM. duo2 was mapped to chromosome 5 between SSLP markers nga106 (33.35 cM) and nga76 (68.40 cM) at position 53.68 ± 0.02 cM.
duo pollen Mutants Possess a Single Generative-Like Nucleus
Mature pollen grains from heterozygous duo1 and duo2 mutants appeared similar in size and appearance to wild-type pollen, but approximately 50% of the population possessed only 2 nuclei (Fig. 1, A and BFigure 1.). Both duo mutants were fully penetrant, and the proportion of mutant pollen did not vary significantly in plants grown in different environments (data not shown). Mutant pollen contained one nucleus with diffuse DAPI staining, typical of the vegetative nucleus, and a second smaller nucleus with more condensed chromatin, similar to the generative nucleus in wild-type pollen. However, careful examination revealed that duo1 and duo2 had distinct nuclear morphologies. In mutant duo1 pollen, the generative-like nucleus always appeared rounded, less compact than duo2 with some heterochromatic regions similar to sperm cells of wild-type pollen (Fig. 1, D and EFigure 1.). In contrast, mutant generative-like nuclei in duo2 were highly compact, and irregular groups of condensed chromosomes with a mitotic morphology were commonly observed (Fig. 1FFigure 1.).
Vegetative cell development and viability was not affected in mutant duo pollen since duo heterozygotes showed 94% to 98% viable pollen based on fluorescein diacetate staining (n > 400). The developmental fate of the vegetative cell was monitored by crossing duo mutants with plants expressing the vegetative nucleus reporter lat52-gus/nia (Twell, 1992). In both mutants, only the vegetative nucleus showed Escherichia coli β-glucuronidase staining, indicating that vegetative cell fate is maintained in the absence of generative cell division (Fig. 1, G and HFigure 1.).
duo pollen Deviates from Wild-Type Development at PMII
To determine when mutant duo pollen deviated from the normal pathway of development, we examined DAPI-stained spores released from different bud stages by light and fluorescence microscopy. No abnormalities were observed during microspore development or during asymmetric division at PMI, and internalized generative nuclei in duo mutants were similar to the wild-type in buds up to −6 stage (data not shown). In wild-type buds at −5 and −4 stages, pollen grains at different phases of generative cell mitosis were observed along with bicellular and tricellular pollen. The percentage of tricellular pollen was therefore used as a measure of developmental age. In the wild type, approximately 24% and 75% of tricellular pollen grains were observed at −5 and −4 bud stages, respectively, and in the succeeding stages, 100% of the pollen population became tricellular (Fig. 2Figure 2.). The number of tricellular pollen grains in duo1 was reduced to 14% and 51% at the −5 and −4 stages. Similarly, in duo2, the percentage of tricellular pollen was reduced to 16% and 49% at the respective stages. We conclude that duo mutations do not affect earlier division at PMI but act specifically to prevent division of the generative cell at pollen mitosis II (PMII).
Figure 2.
Figure 2.
Figure 2.
Developmental analysis of wild-type and duo mutants. Graph showing the percentage of tricellular pollen at different developmental stages in the wild type and in mutant. Shaded box indicates the bud stages at which over 95% of the mitotic figures at PMII (more ...)
Mutant Pollen Grains in duo1 and duo2 Are Bicellular
To determine whether mutant duo pollen grains are binucleate or bicellular, we examined ultrathin sections by transmission electron microscopy. In both duo mutants, two intact membranes around the generative cell demonstrated that both are bicellular (Supplemental Fig. 1, D–F). Other features of the vegetative cell cytoplasm in duo mutants appeared similar to the wild type.
Wild-Type Generative Cell Development and Mitosis
To understand in more detail the failure of generative cell division in the duo mutants, it was first necessary to define the composition and nuclear morphology of spores throughout male germ line development in wild-type plants. We analyzed eight successive bud stages based on their arrangement on the inflorescence axis. At −8 stage, the generative cell (GC) is cut off at the pollen wall or recently internalized (GC early interphase). At −7 and −6 stages, all GCs are internalized and appear rounded (GC late interphase). PMII is not truly synchronous, and the large majority (>95%) of mitotic figures were observed in −5 and −4 bud stages (Fig. 3, A and DFigure 3.). Moreover, pollen with different nuclear morphologies was observed in single anthers. Four distinct classes of pollen grain were scored: rounded generative nucleus, elongated generative nucleus, generative nucleus in mitosis, and two sperm nuclei (Fig. 3AFigure 3.). Although the frequency of each class varied between inflorescences, there was a clear progression of rounded elongated generative nuclei that preceded entry into mitosis (GC prior to PMII). By −3 stage (sperm prior to dehiscence), uniform populations were observed with 2 sperm cell nuclei (Figs. 3AFigure 3. and and4I)4IFigure 4.) that were elongated by +1 stage (sperm at anthesis; see Fig. 1, B and CFigure 1.).
Figure 3.
Figure 3.
Figure 3.
Pollen composition in buds of wild-type and duo mutants at different developmental stages. (A) to (C) show the compositions of pollen populations in individual buds at −6 stage (prior to PMII), at −5 and −4 stages (during PMII), (more ...)
Figure 4.
Figure 4.
Figure 4.
Morphology of generative nuclei and mitotic progression of wild-type pollen stained with DAPI. A, Elongated generative nucleus at late interphase. B, Early prophase with thread-like chromosomes, C, Late prophase with condensed chromosomes. D and E, Metaphase (more ...)
Generative Nuclei in duo1 Do Not Elongate and Enter PMII
We analyzed bud stages −6 to −3 in heterozygous duo1 and reasoned that if the generative cell is arrested before PMII, we should observe a 50% reduction in mitotic figures compared to wild type. Homogeneous pollen populations with rounded generative nuclei were present in −6 stage buds. In succeeding bud stages (−5 and −4), the frequencies of pollen with elongated generative nuclei and those in mitosis were reduced to approximately one-half of those observed in the wild type. Subsequently, an equal proportion of wild-type and mutant pollen grains were present in −3 stage buds (Fig. 3BFigure 3.). We conclude that mutant generative nuclei in duo1 do not elongate and subsequently fail to enter mitosis.
Generative Nuclei in duo2 Complete Morphogenesis and Arrest during Mitosis
In duo2, the composition of pollen populations in bud stages −6 to −4 pollen followed the same overall pattern as wild type. There was no reduction in the proportion of pollen with elongated generative nuclei. Moreover, we observed almost the same proportion of generative nuclei in mitosis in duo2 and wild type. However, by −3 stage, the ratio of wild-type:mutant pollen was approximately 1:1 (Fig. 3CFigure 3.). Therefore, generative nuclei in duo2 undergo normal morphogenesis by elongation and enter mitosis but fail to complete division.
duo1 Fails to Enter PMII whereas duo2 Fails at Prometaphase
To establish a reference for the analysis of mitotic defects in duo1 and duo2, the frequency and progression of mitotic stages at PMII were determined in wild-type plants. Elongation of the generative nucleus preceded entry into mitosis (Fig. 4AFigure 4.). During mitotic prophase, chromatin condenses into thread-like structures (Fig. 4BFigure 4.) that condense further into five compact chromosomes (Fig. 4CFigure 4.). Highly condensed chromosomes congressed on a plane were scored as metaphase (Fig. 4, D and EFigure 4.), and two groups of chromosomes were scored as anaphase (Fig. 4FFigure 4.). At telophase, two sets of congregated chromosomes are well separated (Fig. 4GFigure 4.). Newly formed sperm nuclei are initially round, and chromosomes start to decondense (Fig. 4HFigure 4.). Subsequently, sperm cell nuclei undergo further decondensation and begin to elongate (Fig. 4IFigure 4.). The overall frequency of mitotic figures observed for −5 and −4 bud stages in wild type was 8.4% (n = 3,000 pollen; Fig. 3DFigure 3.).
In heterozygous duo1 plants, generative nuclei at early bicellular stage showed the same morphology as the wild type (Fig. 5AFigure 5.). Throughout PMII (−5 and −4 stages) and in the succeeding stages (−3 to +1 stages), undivided generative nuclei remained unaltered besides an increase in the DAPI fluorescence (Fig. 5, B and CFigure 5.). The overall mitotic index for duo1 was 4.1% (n > 2,500), almost one-half the mitotic index of the wild type, confirming that duo1 fails to enter mitosis.
Figure 5.
Figure 5.
Figure 5.
Morphology of generative nuclei and mitotic progression in duo mutants. A to C, Morphology of DAPI-stained generative nuclei in duo1. Rounded generative nuclei are present in buds before (A), during (B), and after (C) PMII. D to G, Morphology of generative (more ...)
In heterozygous duo2 plants, generative nuclei initially appeared rounded and then elongated prior to mitosis as in the wild type (Fig. 5, D and EFigure 5.). Abnormal mitotic figures were observed in which the chromosomes congressed but were not regularly aligned (Fig. 5, F and GFigure 5.) or formed highly compact structures (Fig. 5HFigure 5.). In the succeeding stages (−3 to +1 stages), chromatin in mutant generative nuclei remained compact but appeared less condensed than during mitosis (Fig. 5IFigure 5.). The overall mitotic index in duo2 was 10.4% (n > 3,000), which was higher than the mitotic index of wild type. This resulted from increases in the frequency of generative nuclei at prophase and prometaphase (Fig. 3DFigure 3.) and could arise if mutant duo pollen spends longer or arrests during these steps of the cell cycle. This is consistent with the reduced number of generative nuclei at anaphase observed in duo2 compared to wild type. We conclude that duo2 enters mitosis but arrests at prometaphase without chromatid separation.
duo1 Bypasses Mitosis and Initiates an Endocycle
In both duo mutants, the generative nucleus in mature pollen was more intensely stained with DAPI than nuclei of wild-type sperm cells, indicating that the generative nucleus has completed DNA replication but failed to divide. This was confirmed by measuring the nuclear DNA contents throughout male germ line development. For reference, the DNA content of telophase nuclei was defined as 1C (sperm nuclei newly formed; Fig. 6AFigure 6.).
Figure 6.
Figure 6.
Figure 6.
DNA content in generative and sperm cell nuclei in wild-type and duo mutants. Bar charts show mean relative DNA contents (DAPI fluorescence values) in the generative nuclei and sperm cell nuclei in wild type (A), duo1 (B), and duo2 (C). The mean C values (more ...)
Wild-type generative nuclei at early interphase had a mean DNA content of 1.15C that increased to 1.74C at the next ontogenetic stage. Prior to PMII, generative nuclei produced mean fluorescence values corresponding to 1.95C. Sperm cell nuclei had a C value of 1.09C at interphase that increased to 1.19C prior to dehiscence and 1.20C at anthesis (Fig. 6AFigure 6.). DNA contents increased significantly at each ontogenetic stage according to one-tailed t tests assuming unequal variances. These data also confirmed earlier findings that the DNA content of the sperm cell nuclei increase progressively after PMII and that sperm cell G1 stage is brief or nonexistent (Friedman, 1999).
In duo1, generative nuclei at early interphase produced a mean fluorescence value that corresponded to 1.08C (Fig. 6BFigure 6.). In buds at late interphase, the DNA content increased to 1.68C, and immediately prior to PMII, generative nuclei had mean DNA content of 1.99C. After PMII, the DNA content of mutant generative cells increased to 2.36C and to 2.46 just prior to anthesis (Fig. 6BFigure 6.). These values were significantly greater than the 2C values measured in generative nuclei just prior to PMII. Therefore mutant generative cell nuclei in duo1 continue S phase during pollen maturation, similar to the continued DNA replication that occurs in wild-type sperm cells (Fig. 6AFigure 6.). In duo2, the mean DNA content of generative nuclei increased during early and late interphase to reach 1.94C immediately prior to PMII. Subsequently, the DNA content of mutant generative nuclei remained constant until anthesis, consistent with the arrest of mutant generative nuclei in mitosis (Fig. 6BFigure 6.).
In summary, our results are consistent with distinct defects in cell cycle progression in duo1 and duo2 mutants compared with wild type (summarized in Fig. 7Figure 7.). Mutant generative cells in duo1 complete S phase but bypass mitosis and continue DNA synthesis before anthesis. However, mutant generative cells in duo2 complete S phase, enter PMII, but fail to exit mitosis as a result of metaphase-anaphase arrest.
Figure 7.
Figure 7.
Figure 7.
Schematic of cell cycle progression in the wild-type and duo mutants. In wild type the generative cell completes DNA replication, enters PMII, and forms the two sperm cells, which re-enter S phase before anthesis. Mutant generative cells in duo1 complete (more ...)
DISCUSSION
DUO Genes as Male Germ Line-Specific Regulators of Cell Cycle Progression
Arabidopsis duo1 and duo2 represent a novel class of male-specific gametophytic mutants that fail to achieve generative cell division. These mutants are distinct from other gametophytic mutants that also fail at PMII, such as gaMS-1 and gaMS-2 in maize (Sari-Gorla et al., 1996, 1997) and mad2 and mad3 in Arabidopsis (Grini et al., 1999), that show pleiotropic phenotypes including reduced pollen size, aborted pollen, and/or cell wall defects. Defects in duo1 and duo2 are restricted to progression of the generative cell cycle. Based on our analysis of viability, ultrastructure and expression of vegetative cell-specific markers, asymmetric division at PMI, and vegetative cell maturation are orchestrated as in wild type. The specific failure of duo1 and duo2 mutants during the generative cell cycle suggests that different sets of genes may be involved in these two postmeiotic divisions. However, it is possible that gene products inherited through meiosis could mask any effects on PMI.
In higher eukaryotes, progression into mitosis is mediated by mitosis promoting factors that consist minimally of CDK and a B-type cyclin regulatory subunit (Ohi and Gould, 1999; Meszaros et al., 2000; Porceddu et al., 2001). We propose that key cell cycle regulatory proteins or factors that modulate them might be impaired in duo1 and duo2. DUO1 and DUO2 appear to function as cell lineage-specific regulators of cell cycle progression; however, DUO genes could also function in other cell divisions including PMI if redundant genes not expressed in the male germ line are able to mask their loss of function.
DUO Genes and Generative Cell Fate Determination
Determination of generative cell fate depends on division asymmetry at PMI (Eady et al., 1995). Moreover, the mechanism of cell fate determination is proposed to involve the unequal distribution of unknown factors in the microspore (Eady et al., 1995; Twell et al., 1998). In duo1 and duo2, asymmetric division at PMI occurs normally, but generative cells are not competent to divide. Therefore, DUO genes could encode cell fate determinants that segregate into the generative cell at PMI to allow generative cell cycle progression. Alternatively, DUO genes could act downstream of generative cell determinants to control cell cycle-specific events during male germ line development. DUO2 appears to act specifically during PMII and is likely to act downstream of generative cell determinants. However, DUO1, which is required for both nuclear morphogenesis and mitotic entry, appears to have a wider and potentially determinative role in generative cell development.
Male Gametic Cells Follow a Simple S-M Cell Cycle
Arabidopsis sperm cells enter S phase immediately after inception and continue DNA synthesis during pollen maturation (Friedman, 1999). We obtained similar results for sperm cells and further demonstrated a progressive increase in the DNA content of generative nuclei. Collectively, these data suggest that generative and sperm cells in Arabidopsis follow a simple S-M cycle and that gap phases are minimal. Although there have been limited studies of generative cell cycle progression, in Capsicum annuum DNA replication does not start immediately after inception but is initiated early, during generative cell detachment (Gonzalez-Melendi et al., 2000). The rapid S-M cycles in Arabidopsis may result from a requirement to limit growth of the gametic cells within the vegetative cell cytoplasm, similar to the alternating S and M phases that characterize cleavage during early embryogenesis in animal systems. In both duo mutants, the DNA content of mutant generative nuclei reached approximately 2C just prior to PMII, suggesting that mutant generative cells complete DNA synthesis normally. In plants, progression into S phase requires the concerted action of cyclin-CDK complexes on specific targets, such as the retinoblastoma/E2F pathways (Gutierrez et al., 2002). Recent data have revealed a negative regulatory role for the Arabidopsis retinoblastoma-related protein, RBR1, on nuclear proliferation in the embryo sac (Ebel et al., 2004). Microarray analysis demonstrated elevated expression of RBR1 in microspores and bicellular pollen (Ebel et al., 2004; Honys and Twell, 2004). Moreover, severe effects on male transmission were observed in RBR1 knockout plants, but potential cell cycle defects remain to be characterized.
Mutant Generative Cells in duo1 Enter an Endocycle
In duo1, mutant generative nuclei continue S phase during pollen maturation to reach approximately 2.5C at anthesis. Therefore the generative cell cycle in duo1 is modified to an endocycle. In Arabidopsis, most tissues except meristems and inflorescence tissues are endoreduplicated (Galbraith et al., 1991). Studies in Drosophila embryos, rat placental trophoblasts, maize endosperm, and tomato fruit (Lycopersicon esculentum) suggest that endoreduplication occurs due to a down-regulation of mitosis promoting factor activity and up-regulation of S-phase CDK activity (Grafi and Larkins, 1995; Sauer et al., 1995; MacAuley et al., 1998; Joubes et al., 1999). We propose that DNA synthesis licensing controls operate in duo1, but in the absence of PMII, which could involve suppression of mitotic cyclin-CDK activity, S-phase specific controls are reactivated, allowing the mutant generative cells to continue S phase.
duo2 Is Arrested in M Phase of the Cell Cycle
The increase in prophase and prometaphase figures in duo2 compared to wild type argues that the mutant generative nuclei spend longer at these phases of mitosis. Moreover, abnormal metaphase figures and the reduced frequency of anaphase figures indicate that the generative nuclei in duo2 arrest at prometaphase and fail to initiate chromosome separation. It is possible that generative nuclei in duo2 undergo premature condensation, leading to arrest at prometaphase. In alfalfa (Medicago sativa) cultured cells, normal chromosome condensation and mitotic progression are dependent on protein phosphatases, PP1 and PP2A. Their inhibition results in hypercondensation of late prophase chromosomes that cannot progress to metaphase (Ayaydin et al., 2000). In this context, we speculate that DUO2 could function as a counter-regulatory component of mitotic kinase activity.
A Potential Role for DUO1 Orthologs in Heterochrony and the Origin of Tricellular Pollen
The developmental alterations in sperm cell formation in the duo mutants are of special interest both to evolutionary and developmental biologists. The development shift of the timing of generative cell division is regarded as an important event that has resulted in the repeated evolution of tricellular species from bicellular species in independent evolutionary clades (Brewbaker, 1967; Friedman, 1999). In angiosperms, heterochronic alterations with respect to S-phase progression and cell cycle activity have led to the diversification of patterns of male gametogenesis (Friedman, 1999 and references therein). DUO1 as a regulator of mitotic entry could provide a potential target for heterochronic mutations. In species with bicellular pollen, DUO1 orthologs would be expressed in the generative cell during pollen tube growth leading to mitotic entry. However, heterochronic mutations resulting in the premature expression of DUO1 orthologs would have the potential to induce generative cell division before anthesis and the production of apomorphic tricellular pollen.
CONCLUSION
This study has provided new insights into the genetic and cytological events associated with male germ line development in Arabidopsis. DUO1 is required for entry into mitosis and could represent a direct or indirect regulator of cyclin-CDK activity or a novel component required for generative cell fate determination. DUO2 is required for mitotic progression and could encode a regulatory component of the mitotic apparatus involved in prometaphase to anaphase transition. The distinctive phenotypes and penetrance of duo1 and duo2 mutations will facilitate cloning of their respective genes to reveal their identities and mechanisms of action during male germ line development.
MATERIALS AND METHODS
Mutant Screen and Growth Conditions
Plant growth conditions and screening of the ethyl methanesulfonate mutagenized population by DAPI staining of pollen were carried out as described previously (Park et al., 1998).
Genetic Analysis
Transmission of mutant alleles was determined through reciprocal testcrosses of heterozygous duo and wild-type No-0 plants, scoring the pollen phenotype of progeny by DAPI staining. Tetrad analysis was performed as described previously (Park et al., 1998). Tetraploid sectors on duo heterozygotes were generated as modified from Vizir and Mulligan (1999). Imbibed seeds from selfed duo heterozygotes were stratified for 3 d at 4°C, washed in 0.1 m potassium phosphate buffer, pH 5.8, and incubated in 2% colchicine for 26 h at room temperature in the dark. The first five flowers from primary inflorescences were screened for pollen showing a significant increase in size. The proportion of pollen showing mutant (duo) and wild-type phenotypes was scored after DAPI staining. Vegetative cell fate was monitored by crossing heterozygous duo plants with a LAT52-GUS-NIa reporter line (Twell, 1992).
duo mutations were independently mapped in F2 populations after outcrossing heterozygous duo plants to wild-type Col-0. DNA was isolated from leaves of over 100 wild-type F2 plants for each mutant to detect linkage. Two to three leaves were collected in 1.5-mL microfuge tubes and frozen in liquid nitrogen. DNA was extracted according to Edwards et al. (1991), but initial homogenization was done with a Silamet dental amalgam mixer (Ivoclar-Viadent, Leicester, UK) for 10 s after the addition of 425 to 600 microns glass beads (acid washed; Sigma-Aldrich, St. Louis). Mapping was carried out using molecular markers showing polymorphism between No-0 and Col-0 using information available at The Arabidopsis Information Resource (TAIR). Chromosome 3 SSLP marker RPF24 located on bacterial artificial chromosome clone F24G16 was amplified with forward (5′-AACAAGTAAAGAAAGTTGAGATTCG-3′) and reverse (5′-CATATGTTTACCAGTCATTGAAACG-3′) primers that showed polymorphism between Col-0 (175 bp) and No-0 (159 bp).
Cytological, Developmental, and DNA Content Analyses
The relationship between flower bud and pollen developmental stages was determined based on the position of buds on the floral axis, with the first open flower termed +1, the first unopened bud termed −1 stage, and progressively younger buds −2 stage and so on (Lalanne and Twell, 2002). Buds were fixed in 3:1 ethanol:acetic acid for 24 h and stored in 75% ethanol at 4°C. Anthers were dissected to release pollen into DAPI staining solution (0.1 m sodium phosphate, pH 7, 1 mm EDTA, 0.1% Triton X-100, 0.4 μg mL−1 DAPI; high grade; Sigma) for 30 min in the dark. A coverslip was mounted, gently squashed to flatten the samples, and sealed with nail varnish. Specimens were viewed and images captured using a CCD camera (KY-F55B; JVC, London) as described previously (Park et al., 1998).
Relative nuclear DNA contents were determined from DAPI fluorescence. Relative fluorescence values were recorded with a fixed exposure and area of interest using Open lab 3.1 (Improvision, Coventry, UK). A net value for each nucleus was calculated after subtraction of a corresponding background reading taken from the cortical cytoplasm. Fluorescence was normalized by comparison with sperm nuclei at telophase that possess 1C DNA content. A one-tailed t test (Excel software; Microsoft, Mountain View, CA) assuming unequal variances was applied to determine whether DNA contents of generative and sperm cell nuclei were significantly different in successive ontogenetic stages.
Ultrastructural Analysis
Materials for ultrathin sectioning were prepared as previously described by Park et al. (1998). Ultrathin sections were viewed with a transmission electron microscope (100CX; JEOL, Tokyo) calibrated using a shadowcast carbon replica of diffraction line gratings with spacing 462.9 nm (Agar Scientific, Stansted, UK).
Supplementary Material
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Acknowledgments
We thank Graham Benskin for providing greenhouse support, James Moore and Ueli Grossniklaus for advice on the Silamet DNA extraction method, and Stefan Hyman and Natalie Allcock for assistance and advice with electron microscopy.
Notes
1This work was supported by the Biotechnology and Biological Sciences Research Council and by the Department of Biology, University of Leicester, UK.
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