74 Growth and Developmen with the vegetative nucleus.contains plastids and pre both embrvogenesis and endost r formation.Prefer ferentially fertilizes the egg cell(16/17)49.As the male ence in fertilization targets would not be critical in thi uni s generally in flowering plants cas plants with isomorphic sperm cells such has zinc-finger transeription factor WRKY2 1581.An increase been an issue of debate. ferential he sperm cells have been inte lized with each wild-type sperm cell.suggesting that n cells has been reported.Knowing whether a sperm cel A pem-like sc has appropriare for both embryo D)mutant The twa ntral easons why the functio developmental programs after fusion with each targer and its T-DNA mutant gonclhusionsln advances have enabled us to same manner 53.Ho a single sperm-l efpnihomal nhi tha A gu live-cell imaging and molecular genetic analysis in A (Figure 3h) the 0 the approach the n sm between the two male gametes and the two female Furthe Additionally the fert cells 6n cen or gam the front (associated with the leading vegetative nuleus ion analyses am ong each gametophytic cel and back sperm cell in the male gemm uni had the s eidentifying new genes involved in egg cel will clucidate the lasti target was observed betwe een the front and back sperm cells accurately fertilize different partners. cells of Arabidopsis This result was consistent with no preference for fusion. Acknowledgements which was observed in most previous mutant analyse RPENSOR.which cells ar essed equally of ere 1. 8S尚器aown Current Opinion in Plant Biology 012.15:70-77 irect.com with the vegetative nucleus, contains plastids and pre￾ferentially fertilizes the egg cell (16/17) [49]. As the male germ unit is generally observed in flowering plants, whether such a preferential fertilization occurs in usual plants with isomorphic sperm cells such as Arabidopsis has been an issue of debate. Emerging data based on an analysis of A. thaliana mutants are complicated and have not clarified the issue of pre￾ferential fertilization. For example, two egg cells in an eostre and rbr1 mutant female gametophyte can be ferti￾lized with each wild-type sperm cell, suggesting that no sperm cell fertilizes only the central cell (Figure 3e) [43,50]. A single sperm-like cell of multicopy suppressor of ira1 and cyclin-dependent kinase a1 (cdka;1) mutants showssingle fertilization with either the egg or the central cell at an equal frequency (Figure 3g) [51,52]. An F-box protein of Arabidopsis, F-box-like 17 (FBL17), targets degradation of cyclin-dependent kinase A1 inhibitors, specifically in male germ cells, and its T-DNA mutant shows a similar impaired seed formation phenotype as that of cdka;1, possibly due to single fertilization in the same manner [53]. However, a single sperm-like cell formed by translational inhibition of sperm cells that express the diphtheria toxin A subunit is likely to cause single fertilization preferentially with the central cell (Figure 3h) [54]. Defects in the regulation of a sperm-specific cis-nat-siRNA of kokopelli (kpl) mutant also result in single fertilization, wherein one of two sperm cells fertilizes either the egg or central cell (Figure 3i) [55]. These discussions about the fertilization capacity of two sperm cells were based on mutant analyses, and therefore, a wild-type analysis is expected. Using a photo-convertible fluorescent protein, mono￾meric Kikume Green-Red, nuclei of two isomorphic Arabidopsis sperm cells were differentially labeled in the pollen tube [11]. Double fertilization of these differently colored wild-type sperm cells revealed that the front (associated with the leading vegetative nucleus) and back sperm cells in the male germ unit had the same opportunity to fertilize the egg cell and the central cell (Figure 3c). That is, no preference in the fertilization target was observed between the front and back sperm cells of Arabidopsis. This result was consistent with no preference for fusion, which was observed in most previous mutant analyses, and no gene was identified as preferentially expressed in one of the two Arabidopsis sperm cells. For example, SHORT SUSPENSOR, which is necessary for regulating unequal divisions of the zygote, is transcribed in both sperm cells [56 ]. Homologs of genes distributed unequally in Plumbago sperm cells are expressed equally in both sperm cells of Arabidopsis [57 ]. One may con￾ceivably assume that isomorphic sperm cells of flowering plants are functionally identical and can be involved in both embryogenesis and endosperm formation. Prefer￾ence in fertilization targets would not be critical in this case. Another gene expressed in the male gametophyte to establish polarity in the zygote has been identified as the zinc-finger transcription factor WRKY2 [58 ]. An increase in the number of such genes will provide insights into the capacity of the two sperm cells. Additionally, epigenetic regulation and reprogramming of the sperm cells have been intensively studied [6,59–63]. No epigenetic regulation specific to one of the two sperm cells has been reported. Knowing whether a sperm cell has appropriate epigenetic regulation for both embryo￾genesis and endosperm formation would be of interest. The two male chromatins undergo distinct epigenetic reprogramming (chromatin remodeling) after fertilization [59]. This might be one of the reasons why the function￾ally identical sperm cells might contribute to different developmental programs after fusion with each target. Conclusions Recent live-cell imaging advances have enabled us to visualize male and female gamete interactions during double fertilization. The three-step behavior of sperm cells during double fertilization is now apparent. Both live-cell imaging and molecular genetic analysis in Ara￾bidopsis support the idea that the two sperm cells have an equal ability to fertilize each female gamete. Rapid blocking in both the egg and central cell might be critical to avoid mis-targeting of two identical sperm cells. To approach the mechanism of double fertilization, further insights are required into the cell-to-cell communications between the two male gametes and the two female gametes during the 7.4-min immobility phase. Further development of imaging techniques will provide power￾ful tools to gain higher temporal and spatial-resolution information. Additionally, novel approaches including visible screening using fluorescent marker lines of game￾tophytic cells [64] and large-scale transcriptome and gene-expression analyses among each gametophytic cell [65–68] will accelerate identifying new genes involved in double fertilization. In the near future, these new insights will elucidate the long-lasting issue of how two sperm cells accurately fertilize different partners. Acknowledgements We thank Taeko Sasaki for illustration of Figure 3b. Y.H. was supported by a grant (number 9138) from the Japan Society for the Promotion of Science Fellowships and by GCOE program (Nagoya Univeristy). References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1. Higashiyama T: The synergid cell: attractor and acceptor of the pollen tube for double fertilization. J Plant Res 2002, 115:149-160. 74 Growth and Development Current Opinion in Plant Biology 2012, 15:70–77 www.sciencedirect.com