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First published online December 23, 2004; 10.1104/pp.104.053165 Plant Physiology 137:297-307 (2005) © 2005 American Society of Plant Biologists Male Germ Line Development in Arabidopsis. duo pollen Mutants Reveal Gametophytic Regulators of Generative Cell Cycle Progression1,[w]Department of Biology, University of Leicester, Leicester LE1 7RH, United Kingdom
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
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
Despite the wide range of studies on cell cycle regulation in plants (for review, see Stals and Inze, 2001
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
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
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.
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 B). 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 E). 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. 1F).
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
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. 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).
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.
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 D). 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. 3A). 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. 3A and 4I) that were elongated by +1 stage (sperm at anthesis; see Fig. 1, B and C).
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. 3B). We conclude that mutant generative nuclei in duo1 do not elongate and subsequently fail to enter 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. 3C). Therefore, generative nuclei in duo2 undergo normal morphogenesis by elongation and enter mitosis but fail to complete division.
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. 4A). During mitotic prophase, chromatin condenses into thread-like structures (Fig. 4B) that condense further into five compact chromosomes (Fig. 4C). Highly condensed chromosomes congressed on a plane were scored as metaphase (Fig. 4, D and E), and two groups of chromosomes were scored as anaphase (Fig. 4F). At telophase, two sets of congregated chromosomes are well separated (Fig. 4G). Newly formed sperm nuclei are initially round, and chromosomes start to decondense (Fig. 4H). Subsequently, sperm cell nuclei undergo further decondensation and begin to elongate (Fig. 4I). The overall frequency of mitotic figures observed for –5 and –4 bud stages in wild type was 8.4% (n = 3,000 pollen; Fig. 3D). In heterozygous duo1 plants, generative nuclei at early bicellular stage showed the same morphology as the wild type (Fig. 5A). 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 C). 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.
In heterozygous duo2 plants, generative nuclei initially appeared rounded and then elongated prior to mitosis as in the wild type (Fig. 5, D and E). Abnormal mitotic figures were observed in which the chromosomes congressed but were not regularly aligned (Fig. 5, F and G) or formed highly compact structures (Fig. 5H). In the succeeding stages (–3 to +1 stages), chromatin in mutant generative nuclei remained compact but appeared less condensed than during mitosis (Fig. 5I). 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. 3D) 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.
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. 6A).
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. 6A). 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. 6B). 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. 6B). 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. 6A). 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. 6B). 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. 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.
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
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
Determination of generative cell fate depends on division asymmetry at PMI (Eady et al., 1995
Arabidopsis sperm cells enter S phase immediately after inception and continue DNA synthesis during pollen maturation (Friedman, 1999
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
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
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
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.
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
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
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)
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 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.
Materials for ultrathin sectioning were prepared as previously described by Park et al. (1998)
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. Received September 9, 2004; returned for revision November 13, 2004; accepted November 15, 2004.
1 This work was supported by the Biotechnology and Biological Sciences Research Council and by the Department of Biology, University of Leicester, UK. 
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.053165. * Corresponding author; e-mail twe{at}le.ac.uk; fax 44–(0)–116–252–2791.
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ABSTRACT
RESULTS
) in tetraploids that range from 0.0 to 0.3 (Butruille and Boiteux, 2000
-glucuronidase staining, indicating that vegetative cell fate is maintained in the absence of generative cell division (
