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The production of two functional sperm cells within each male gametophyte is essential for double fertilization in flowering plants and involves a single mitotic division of the male germ cell and cell specification to produce functional gametes. Several proteins that are important regulators of male germ cell division have been identified as well as the R2R3 MYB protein DUO1 that has a dual role in cell division and cell specification. We recently identified a novel regulatory protein DUO3, that has overlapping roles with DUO1 in cell division and specification and shows similarity to GON4 related cell lineage regulators in animals. DUO3 also has important roles outside the germline and is required for embryo patterning and meristem function. We outline the regulatory roles of DUO3 in male germline development and its possible mechanisms of action as a lineage regulator in current models that link germ cell cycle control and gamete specification.
The two sperm cells required for double fertilization in flowering plants are produced after an asymmetric division of the haploid microspore produces a large vegetative cell and a smaller germ cell, thereby establishing the male germline (reviewed in ref. 1; Fig. 1A). The germ cell is engulfed within the vegetative cell cytoplasm where it divides to produce the two sperm cells. The germ cell also goes through a process of specification, with ~6,000 genes expressed in sperm cells,2 many of which show specific or enhanced expression in the male germline and/or are essential for fertilization.2–4 Since 2005 a number of proteins with important regulatory roles in either germ cell division5–9 or both germ cell division and specification10–12 have been described, enabling the formulation of basic models for the regulation of male germline development.12,13 In our recent publication14 we identify a novel regulatory protein, DUO POLLEN3 (DUO3) that has essential roles in germ cell division and specification, as well as vital sporophytic functions. Here we present the role of DUO3 in an emerging model for the regulation of male germline development in Arabidopsis (Fig. 1B) and briefly discuss the wider role and possible mechanism of DUO3 function.
DUO3 has a post-meiotic role in germ cell division, with the majority of mutant duo3 germ cells failing to divide by anthesis, resulting in pollen with a single germ cell rather than two sperm cells. Although a similar phenotype is observed in other mutants, the role of DUO3 appears different (Fig. 1B). After asymmetric microspore division the newly formed germ cell enters S phase which requires the core cell cycle regulator CDKA.5,6 Shortly after asymmetric division CDKA is inactive due to the presence of the inhibitory proteins KRP6 and KRP7. These inhibitors are then targeted by the F-box protein FBL17 that is part of an SCF E3 ubiquitin protein ligase complex that mediates KRP6/7 degradation via the proteosome.7 CDKA forms a complex with CYCD and phosphorylates RBR which relieves repression of the E2F pathway, enabling expression of genes required for progression through S-phase.15 Like duo3 mutant germ cells both cdka and fbl17 mutant germ cells have a single germ cell at anthesis however these germ cells fail to complete a single round of S-phase6,7 while duo3 germ cells complete S-phase at a similar rate to wild type germ cells. Thus DUO3 does not regulate CDKA or other cell cycle regulators, such as CYCD, involved in S phase initiation and progression.
Rather DUO3 appears to have a role in G2/M transition and entry of germ cells into mitosis. The majority of duo3 germ cells fail to divide and those that do show a delay in comparison to wild type germ cells. Undivided duo3 germ cells either remain in G2 (~2C DNA content at anthesis) or skip mitosis and re-enter S-phase (>2C DNA content at anthesis). Mutant duo1 germ cells also complete S phase and then fail to divide,16 and like duo3 germ cells some duo1 germ cells also re-enter S phase despite not passing through mitosis. Germ cells lacking DUO3 however differ from duo1 germ cells in that they express CYCB1;1, an important regulator of the G2/M transition, while undivided duo1 germ cells do not.12 The differing expression of CYCB1;1 may explain why some duo3 but no duo1 germ cells divide. Thus DUO3 regulates G2/M regulatory factors other than CYCB1;1 but which could include other CYCB family members, or their substrates, that are also expressed in pollen17 or the activity of the CYCB1;1 protein itself. The range of phenotypes of duo3 germ cells suggests that DUO3 may regulate multiple targets and that regulation maybe indirect. Interestingly expression of CYCB1;1 in duo1 germ cells only partially restores germ cell division,12 so it is possible that DUO1 and DUO3 may have G2/M targets in common other than CYCB1;1 (dashed line Fig. 1B).
DUO3 also appears to be important for cell cycle progression in sporophytic plant tissues. DUO3 is expressed in regions with enhanced cell division activity and potential, such as meristems and vascular tissue. Furthermore, duo3 embryos are defective in morphogenesis and patterning and show a reduced rate and loss of co-ordination of cell division compared to wild germlinetype embryos. As embryo phenotypes are also variable, DUO3 may also regulate a number of different cell cycle targets in sporophytic tissues.
The single germ cells in cdka and fbl17 pollen are functional and can fertilize the egg cell5–7,9 indicating that these germ cells have been correctly specified, and that the processes of cell cycle progression and gamete specification can be uncoupled. Mutant duo1 and duo3 germ cells however do not fertilize and both DUO1,12 and DUO3 are required for the full expression of male germline markers (Fig. 1B). Thus DUO1 and DUO3 are required for sperm cell specification as well as division and may have a role integrating the two processes (Fig. 1B).
Although both DUO1 and DUO3 are involved in gamete specification they have distinct, but overlapping, roles. DUO1 is required for expression of all three (MGH3, GCS1 and GEX2) germline markers analysed and there is virtually no marker expression in any duo1 germ cells.12 While DUO3 is required for GCS1 expression, it is not required for MGH3 expression and GEX2 expression is highly variable in duo3 germ cells. This may relate to differences in activity of the two proteins. As DUO1 is an R2R3 MYB transcription factor16 it may bind to target promoter regions to directly activate transcription. The variable expression of GEX2 in duo3 germ cells indicates that regulation by DUO3 is likely to be complex and indirect. Interestingly MGH3 (regulated only by DUO1) is strictly male germline specific while GEX2 and GCS1, (regulated by both DUO1 and DUO3) have weak expression levels in other plant tissues. DUO3 may also have a role in specification in sporophytic tissues such as meristem formation as duo3 embryos display morphological defects in the presumptive shoot apical meristem resulting in failure to produce true leaves after germination.
DUO3 is conserved throughout the land plants and DUO3 from moss restores male germline function (both division and specification) in Arabidopsis duo3 germ cells. As moss does not produce pollen and the moss cDNA was isolated from green non-reproductive tissues,18 DUO3 appears to possess general regulatory functions that are conserved in gametophytic and sporophytic tissues. DUO3 may also have a similar molecular function to the Gonadless4-Like (GON4-L) proteins in animals. Not only do DUO3 and GON4-L proteins share sequence similarity, both proteins are located in the nucleus and there are some phenotypic similarities between duo3 and gon4-l mutants.19,20 In Caenorhabditis elegans GON4 is required for normal cell cycle progression in cell lineages involved in gonad formation19 and mutations in the zebrafish GON4-L homolog Ugly Duckling (UDU) result in primitive erythroid cells with impaired proliferation, and incorrect cell specification.20 In udu mutants erythroid cells accumulate and arrest at G2/M similar to duo3 germ cells indicating a link between the molecular functions of DUO3 and GON4-L proteins in cell cycle control.
The molecular mechanism of DUO3 and GON4-L regulation of targets is currently unknown. Although it is possible that DUO3 directly activates targets as part of an activating complex we propose that its function more likely involves chromatin remodeling, preparing DNA for future transcriptional activation that has been proposed for UDU.20 A role in chromatin remodeling is consistent with the likely indirect and complex role of DUO3 dependent activation of cell cycle and specification targets discussed above. As DUO3 and GON4-L proteins are large proteins with a number of potential protein and DNA binding sites, and some GON4-L proteins bind specific proteins,21,22 DUO3 and GON4-L proteins may acts as a scaffolding protein, bringing together a range of proteins at certain sites in the DNA. Proteins recruited by such as complex could include histone modifying enzymes such as histone acetyltransferases that alter the chromatin structure allowing further transcriptional activators, such as DUO1 in the male germ cells of Arabidopsis, access to promoter regions. Interestingly some DUO1 targets, such as MGH3, do not require DUO3 thereby defining different pathways of germline-specific activation (Fig. 1B). Confirming the molecular function of DUO3 proteins and their molecular interactions, along with determining the full range of DUO3 targets remains an important challenge that will help to uncover new features of the mechanisms that link cell specification and cell cycle control in plant development.
Previously published online: www.landesbioscience.com/journals/psb/article/9950