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

Summary

We demonstrate a method to determine successful or failed fertilization on the basis of sperm nuclear morphology in Arabidopsis double fertilization using an epifluorescence microscope.

Abstract

Flowering plants have a unique sexual reproduction system called ‘double fertilization', in which each of the sperm cells precisely fuses with an egg cell or a central cell. Thus, two independent fertilization events take place almost simultaneously. The fertilized egg cell and central cell develop into zygote and endosperm, respectively. Therefore, precise control of double fertilization is essential for the ensuing seed development. Double fertilization occurs in the female gametophyte (embryo sac), which is deeply hidden and covered with thick ovule and ovary tissues. This pistil tissue construction makes observation and analysis of double fertilization quite difficult and has created the present situation in which many questions regarding the mechanism of double fertilization remain unanswered. For the functional evaluation of a potential candidate for fertilization regulator, phenotypic analysis of fertilization is important. To judge the completion of fertilization in Arabidopsis thaliana, the shapes of fluorescence signals labeling sperm nuclei are used as indicators. A sperm cell that fails to fertilize is indicated by a condensed fluorescence signal outside of the female gametes, whereas a sperm cell that successfully fertilizes is indicated by a decondensed signal due to karyogamy with the female gametes’ nucleus. The method described here provides a tool to determine successful or failed fertilization under in vivo conditions.

Introduction

Flowering plants produce seeds through double fertilization, a process that is directly controlled by interactions between proteins localized on gamete plasma membrane^1,2. Flowering plant male gametes, a pair of sperm cells, develop in pollen. A pollen tube that grows after pollination delivers a pair of sperm cells to female gametes, an egg cell and a central cell, which develop in an embryo sac. After the male and female gametes meet, proteins on the gamete surface promote recognition, attachment, and fusion to complete double fertilization. In previous studies, the male gamete membrane proteins GENERATIVE CELL SPECIFIC 1 (GCS1)/HAPLESS2 (HAP2)^3,4 and GAMETE EXPRESSED 2 (GEX2)⁵ were identified as fertilization regulators involved in gamete fusion and attachment, respectively. We recently identified a male gamete-specific membrane protein, DUF679 DOMAIN MEMBRANE PROTEIN 9 (DMP9), as a fertilization regulator involved in gamete interaction. We found that a decrease of DMP9 expression results in significant inhibition of egg cell fertilization during double fertilization in A. thaliana⁶.

As double fertilization occurs in an embryo sac, which is embedded in an ovule that is further wrapped with ovary tissue, it is difficult to observe and analyze the states of double fertilization processes. For this reason, there are still many unclear points that hinder a complete understanding of the whole mechanism of double fertilization control. The establishment of observation techniques to trace the behavior of gametes during double fertilization under in vivo conditions is indispensable for the functional analysis of potential candidates for fertilization regulators. Recent studies have yielded marker lines where gamete subcellular structures are labeled with fluorescent proteins. In this article, we demonstrate a simple and quick protocol for observing double fertilization that has occurred in an embryo sac derived from artificially pollinated pistils. Using sperm cell nucleus marker line HTR10-mRFP⁷, the fertilization state of each female gamete can be discriminated on the basis of sperm nuclear signal morphology. Our protocol focusing on such a morphological change of the sperm nuclei at fertilization can efficiently obtain a sufficient amount of data for statistical proof. A DMP9-knockdown line with HTR10-mRFP background (DMP9^{KD/HTR10-mRFP}) was used as male plants to show a single fertilization pattern. The protocol is also suitable for the functional analysis of other fertilization regulators.

Protocol

1. Artificial Pollination

NOTE: Before starting the process, a pair of No. 5 forceps is required.

1. Grow A. thaliana (Col-0) at 22 °C under a 16-h light/8-h dark cycle in a growth chamber.
   NOTE: Cut and remove the first developed flower stalk with scissors to promote development of axillary buds. Vigorously growing plants (2-3 weeks after cutting of the first stalk; plant height about 25 cm) are suitable for analysis.
2. To emasculate, remove sepal (Figure 1B), petal (Figure 1C), and stamen (Figure 1D) of flower buds at stage 11⁸ (Figure 1A) using No. 5 forceps. Bud with bits of petals seen at the top is best.
   NOTE: Use a suitable female gamete marker line. In this protocol, we used a wild type plant as the female parent.
3. Fifteen to eighteen hours after emasculation, take the stamen of a DMP9^{KD/HTR10-mRFP} flower at stage 13⁸ by pinching the filament with forceps.
4. To pollinate, gently pat the stigma of an emasculated pistil several times with a dehiscent anther.

2. Preparation of Ovule for Observation

NOTE: The following items are required: a slide glass with double-sided tape attached, No. 5 forceps, a 27 G injection needle, and a dissecting microscope.

1. 7 to 8 h after pollination (HAP), collect the pistil and place it on the double-sided tape, then press gently with forceps to fix the pistil on the tape (Figure 2A,A’).
   NOTE: Most ovules in a pistil receive at least one pollen tube 10 HAP⁹. If both or any one of the sperm cells from the first pollen tube fail to fertilize, a second pollen tube would be attracted by the ovule due to the fertilization recovery system¹⁰. To analyze the sperm nuclei morphology from the first pollen tube, it is recommended to complete ovule preparation by 10 HAP at the latest.
2. Cut off the upper and lower ends of the ovary using an injection needle under a dissecting microscope (Figure 2B,B’).
3. Slit the ovary wall along both sides of the replum (Figure 2C,C’) by moving the tip of the injection needle.
   NOTE: Insert the injection needle shallowly to prevent ovule separation from the septum.
4. Evert the ovary wall by using the injection needle (Figure 2D,D’).
5. Pinch the base of the septum to which ovules are connected, and lift it up carefully with forceps (Figure 2E).
6. Transfer the ovules into a drop of water on a slide glass, and gently cover with a cover glass for observation under a fluorescence microscope (Figure 2E,E’).

3. Microscopy

NOTE: In this protocol, we used an epifluorescence microscope equipped with a fluorescence filter cube (see Table of Materials), a digital camera, and the accompanying software.

1. Acquire images of ovules containing sperm nuclei labeled with mRFP using a 20x or 40x objective lens and the equipped digital camera.
2. Confirm the number of mRFP-labeled sperm nuclei in an embryo sac.
   NOTE: Ovules containing two mRFP-labeled sperm nuclei can be included in the population size for statistical analysis.
3. Confirm the shape and position of each mRFP-labeled sperm nucleus in an embryo sac.
   NOTE: Immediately after being released from a pollen tube, a pair of condensed mRFP-labeled sperm nuclei are localized between the egg and the central cell. A decondensed mRFP-labeled sperm nucleus detected at the side of chalazal end indicates central cell fertilization, for instance. By using a suitable female gamete membrane marker line, as shown in Supplementary Figure 1, whether or not the sperm cell is undergoing plasmogamy (after membrane fusion but before karyogamy) can be monitored clearly.

Representative Results

Ovules from a pistil pollinated with DMP9^{KD/HTR10-mRFP} were collected at 7-8 HAP and observed.

Most ovules contained two decondensed mRFP-labeled sperm nuclei at the egg cell (micropylar side) and central cell (charazal end side) nucleus positions, respectively (Figure 3A), indicating successful double fertilization. In addition, ovules containing a decondensed mRFP-labeled sperm nucleus at the central cell nucleus plus a condensed mRFP-labeled sperm nucleus outside the egg cell (Figure 3B) were observed, indicating single fertilization. In the case of failed double fertilization, two condensed mRFP-labeled sperm nuclei were observed at the boundary of the egg cell and the central cell (Figure 3C), as in gcs1 mutant sperm cells (Figure 3D)^3,4.

In the analysis using DMP9^{KD/HTR10-mRFP} pollen, ovules containing a single condensed mRFP-labeled sperm nucleus (Figure 4A) or three or more mRFP-labeled sperm nuclei (Figure 4B) were rarely observed.

Figure 1: Flower buds suitable for emasculation. (A) A flower bud at stage 11, showing bits of the petals at the top. (B-D) A flower bud in which was removed the sepals (B) and petals (C) and that was then emasculated completely (D). The anther dehiscence has not occurred yet. Scale bar = 1 mm. Please click here to view a larger version of this figure.

Figure 2: Flow of ovule sample preparation. The procedures are shown by illustrations (A-E) and photographs (A’-E’). (A, A’) A pistil is placed on double-sided tape attached to a slide glass, and its upper and lower ends are cut off with an injection needle. (B, B’) The ovary wall is incised along both sides of the replum. (C, C’) Both sides of the ovary walls are opened, everted, and pressed onto the tape for fixing. (D, D’) The septum where ovules are connected in arrays is exposed. (E, E’) One end of the septum is pinched and lifted up with forceps. The septum with connecting ovules is transferred to a drop of water on a slide glass. Please click here to view a larger version of this figure.

Figure 3: Fertilization phenotypes judged by sperm nuclear signal morphology in ovule. (A) Successful double fertilization indicated by two decondensed mRFP-labeled sperm nuclei (arrowheads). (B) Single fertilization by DMP9^{KD/HTR10-mRFP} sperm cells indicated by one decondensed mRFP-labeled sperm nucleus (arrowhead) and one condensed mRFP-labeled sperm nucleus (arrow). (C) Failed double fertilization by DMP9^{KD/HTR10-mRFP} sperm cells indicated by two condensed mRFP-labeled sperm nuclei (arrows). (D) An example of failed double fertilization by gcs1 ^HTR10-mRFP sperm cells. A marker line where the egg cell nucleus (EN) is labeled with RFP was used. Two condensed mRFP-labeled sperm nuclei (arrows) are arrested without fertilization at 18 HAP. (E) Schematic illustration of an ovule. EC = egg cell, CC = central cell, SC = synergid cells, ES = embryo sac, MP = micropyle, CHZ = chalazal end. Scale bars = 20 μm. Please click here to view a larger version of this figure.

Figure 4: Other potential fertilization phenotypes. Ovules from a pistil pollinated with DMP9^{KD/HTR10-mRFP} rarely contain a single condensed mRFP-labeled sperm nucleus (A), or three or more condensed mRFP-labeled sperm nuclei (B) (arrows). Scale bars = 20 μm. Please click here to view a larger version of this figure.

Supplementary Figure 1: Condensed sperm nuclei at plasmogamy during fertilization, judged by combination with a female plasma membrane marker line. pFWA::GFP-PIP where the central cell plasma membrane is visualized was used as female parent (GFP). GFP-PIP also labels endo membranes¹². The ovule contains two condensed mRFP-labeled sperm nuclei (arrows; RFP). The RFP signal at charazal end side (arrow) is shown in the central cell (Merge), indicating that the sperm nucleus is in plasmogamy (after gamete membrane fusion, but before karyogamy). The panel on the right is a magnification of the area surrounded by dashed lines in the merged image. A blank area marked by an asterisk corresponds to the position of the central cell nucleus. The dashed line indicates the outline of the central cell facing the egg cell. Scale bar = 20 μm. Please click here to download this file.

Discussion

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.

Materials

Name	Company	Catalog Number	Comments
BX51	Olympus		Epifluorescence microscope
Cover glass	Matsunami glass	C018181
DMP9KD/HTR10-mRFP			Arabidopsis thaliana, HTR10-mRFP background Takahashi et al. (2018)6
Double-sided tape	Nichiban	NW-15S	15 mm width
DP72	Olympus		Degital camera
Forceps	Vigor		Any No. 5 forceps are available
Growth chamber	Nihonika	LPH-411PFQDT-SP
HTR10-mRFP			Arabidopsis thaliana, ecotype Columbia-0 (Col-0) background Ingouff et al. (2007)7
Injection needle	Terumo	NN-2719S	27 G
Slide glass	Matsunami glass	S9443
SZX9	Olympus		Dissecting microscope
U-MRFPHQ	Olympus		Fluorescence Filter Cube (Excitation: BP535-555, Emission: BA570-625, Dichromatic mirror:DM565)
UPlanFL N 40x	Olympus		Objective lens (NA 1.3), oil-immersion
UPlanSApo 20x	Olympus		Objective lens (NA 0.75), dry