Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

HTR10-mRFP labels paternal chromatin (i.e., visualizes sperm cell nuclei), and the dynamics in double fertilization have been reported⁷. Immediately after release from a pollen tube, HTR10-mRFP-labeled sperm nuclei are still condensed. However, each of the sperm nuclei is decondensed upon merging with a fertilized female gamete nucleus at karyogamy three to four hours after gamete membrane fusion⁷. Unfertilized sperm cells remain condensed, as shown in an embryo sac in which gcs1 sperm cells are arrested without fertilization (Figure 3D). Usually, when the fertile HTR10-mRFP is used as the pollen parent, 57.9% ± 17.8% (mean ± s.d.; n = 16 pistils) of ovules in a pistil at 7-8 HAP contain two mRFP-labeled sperm nuclei signals, and almost all of the signals (97.2%) are decondensed, reflecting successful double fertilization (a similar signal pattern is shown in Figure 3A). In the case of DMP9^{KD/HTR10-mRFP}, ovules containing two mRFP-labeled sperm nuclei were observed at a similar frequency to that of HTR10-mRFP, but 17.6% of them showed single fertilization⁶. We adopted the HTR10-mRFP dynamics for observation of a large number of ovules under in vivo conditions using an epifluorescence microscope. The protocol is simple and quick, which enables the collection of sufficient data for statistical proof. Using this protocol as the first instance, the significance of single fertilization of the central cell by DMP9^{KD/HTR10-mRFP} sperm cells was proved⁶.

Based on the morphological differences of HTR10-mRFP-labeled sperm nuclei derived from one pollen tube, the fertilization patterns are classified into three phenotypes: (1) successful double fertilization, which is characterized by two decondensed HTR10-mRFP-labeled sperm nuclei (Figure 3A); (2) single fertilization, which features one condensed HTR10-mRFP-labeled sperm nucleus and one decondensed HTR10-mRFP-labeled sperm nucleus (Figure 3B); and (3) failed double fertilization, which has two condensed HTR10-mRFP-labeled sperm nuclei (Figure 3C).

Other potential causes of phenotypes (2) and (3) should be considered. It has been reported that sperm cells respectively begin plasmogamy with an egg cell or central cell several minutes after being released into an embryo sac under semi-in vitro culture conditions¹¹. In addition, a time lag of a few minutes exists between the first and second fertilizations, although there is no preference for the order of fertilization of the egg cell and the central cell¹¹. Therefore, a pair of condensed and decondensed sperm nuclei in phenotype (2) might indicate a time lag between the fertilizations instead of single fertilization. In order to confirm the occurrence of single fertilization at a significant frequency in phenotype (2), a number of samples sufficient for statistical analysis should be examined. Phenotype (3), which has two condensed sperm nuclei (Figure 3C), could reflect a period of immobility of sperm cells prior to plasma membrane fusion¹² or the period between plasmogamy and karyogamy. Therefore, two condensed sperm nuclei at 7-8 HAP do not fully reflect 'failure' of double fertilization, and it is necessary to observe the ovules at a later time after pollination to assess the success or failure of double fertilization. In this regard, ovule observation at 16-18 HAP is recommended, because most of the ovules would have completed double fertilization with sperm cells delivered by the first pollen tube, and HTR10-mRFP signals of unfertilized sperm nuclei would remain until the next day of pollination, as shown in Figure 3D.

A pattern of single condensed HTR10-mRFP-labeled sperm nucleus (Figure 4A) was rarely detected, which was likely due to the quick disappearance of another paternal HTR10-mRFP signal in the fertilized female gamete as a result of single fertilization. In this analysis, three or more HTR10-mRFP-labeled sperm nuclei were also detected as a rare case (Figure 4B), due to second pollen tube acceptance by the fertilization recovery system¹⁰. In this protocol, these cases were excluded from the data, because tracing the behaviors of two sperm cells derived from one pollen tube is critical for accurate assessment.

Since the polarity for the positions of the female gametes in an embryo sac is well regulated (i.e., the egg cell and the central cell differentiate at the micropylar and the chalazal side, respectively), which female gamete is fertilizing can be judged by the relative positions of the mRFP-labeled sperm nuclei. However, there is a limitation in distinguishing between unfertilized and plasmogamy states, because both states are indicated by the condensed sperm nuclei signal. To evaluate precisely whether plasmogamy after gamete membrane fusion has occurred or not when the sperm nuclei are condensed, it is required to use female gamete membrane marker lines, as reported by Takahashi et al. (2018)⁶. For example, when a pFWA::GFP-PIP plant¹² in which the central cell plasma membrane was visualized was used as a female parent, it is clear that a sperm cell fused with the central cell was in plasmogamy (Supplementary Figure 1).

In summary, our protocol described here can be used for statistical assessment of success or failure of fertilization in each female gamete. Although the employment of the female plasma membrane marker is required to judge the plasmogamy, our method is useful for functional analysis of the fertilization regulator.