Abstract
A putative nuclear lamina protein, KAKU4, modulates nuclear morphology in Arabidopsis thaliana seedlings but its physiological significance is unknown. KAKU4 was strongly expressed in mature pollen grains, each of which has a vegetative cell and two sperm cells. KAKU4 protein was highly abundant on the envelopes of vegetative nuclei (VNs) and less abundant on the envelopes of sperm cell nuclei (SCNs) in pollen grains and elongating pollen tubes. VN is irregularly shaped in wild-type pollen. However, KAKU4 deficiency caused it to become more spherical. These results suggest that the dense accumulation of KAKU4 is responsible for the irregular shape of the VNs. After a pollen grain germinates, the VN and SCNs migrate to the tip of the pollen tube. In the wild type, the VN preceded the SCNs in 91–93% of the pollen tubes, whereas in kaku4 mutants, the VN trailed the SCNs in 39–58% of the pollen tubes. kaku4 pollen was less competitive than wild-type pollen after pollination, although it had an ability to fertilize. Taken together, our results suggest that controlling the nuclear shape in vegetative cells of pollen grains by KAKU4 ensures the orderly migration of the VN and sperm cells in pollen tubes.
Highlight The nuclear envelope protein KAKU4 is involved in controlling the migration order of vegetative nuclei and sperm cells in pollen tubes, affecting the competitive ability of pollen for fertilization.
Introduction
In Arabidopsis thaliana, pollen grains contain a vegetative cell and two sperm cells (Borg and Twell, 2011; McCue et al., 2011). One of the sperm cells fertilizes the egg cell to produce the embryo and the other fertilizes the central cell to produce the endosperm (Hamamura et al., 2011; Kawashima and Berger, 2011; Maruyama et al., 2015). The vegetative nucleus (VN) has irregular shapes (Vogler et al., 2015) and is physically connected to one of the two sperm cells. Together, the VN and the two sperm cells form a functional unit termed the male germ unit (McCue et al., 2011). After pollination, the pollen grain germinates. Then, the VN and its trailing sperm cells move into and migrate along the elongating pollen tube, although they occasionally change their migration order (Zhou and Meier, 2014).
Nuclear movements are driven by the plant-specific motor protein myosin XI-i (Tamura et al., 2013). In A. thaliana, the migration order of the VN and sperm cells is not affected by myosin XI-I deficiency (Zhou and Meier, 2014), but it is affected by the outer nuclear membrane proteins WIPs (WIP1, WIP2, and WIP3) and WITs (WIT1 and WIT2) and the possible inner nuclear membrane proteins SUNs (SUN1 and SUN2) (Zhou et al., 2015; Zhou and Meier, 2014).
Plant nuclei have a fibrillar meshwork lamina structure beneath the inner nuclear membrane (Fiserova et al., 2009). KAKU4 (after the Japanese word for nucleus, kaku) is a candidate component of the plant nuclear lamina (Goto et al., 2014; Meier et al., 2017; Poulet et al., 2017). In epidermal cells of the mature vegetative tissues of A. thaliana, a KAKU4-deificient mutant (kaku4) has smaller spherical-shaped nuclei, compared with spindle-shaped nuclei of the wild type. A spherical phenotype is also found in mutants defective in the outer nuclear membrane proteins WIPs (Zhou et al., 2012), in the possible inner nuclear membrane proteins SUNs (Zhou et al., 2012), and in the nuclear envelope-associated proteins CRWNs (CRWN1 and CRWN4) (Dittmer et al., 2007; Goto et al., 2014; Sakamoto and Takagi, 2013; Wang et al., 2013).
In this study, we show that KAKU4 is highly expressed in the VNs of pollen grains and tubes, resulting in deep invagination of the VN envelope. We propose that KAKU4-mediated deformation of the VN controls the proper migration order of VN and sperm cells during pollen tube growth in A. thaliana.
Materials and methods
Gene expression data from public databases
We assessed the gene expression level data in various tissues deposited in the database Atted-II (http://atted.jp/), and then downloaded the folder “Expression_data_Development” and used the file “AtGE_dev_gcRMA” which contains the raw data of microarray experiments (Schmid et al., 2005). We chose the values of the genes of interest from wild-type plants for graphical presentation.
Plant materials
Arabidopsis thaliana (Columbia-0) was used as the wild-type line. T-DNA insertion mutants (SALK_076754 [kaku4-2], SALK_010298 [kaku4-3], SAIL_711_E09 [kaku4-4], and SALK_041774 [crwn1-2],) and EMS-mutagenized mutants (kaku2 and kaku4-1) were isolated previously (Goto et al., 2014). The transgenic plants ProKAKU4:KAKU4-tRFP kaku4-2 and ProKAKU4:KAKU4-EYFP kaku4-3, in which the genomic fragment of the splice variant KAKU4.2 was expressed, were also used (Goto et al., 2014). Seeds were germinated and grown on MS medium containing 0.5% gellan gum at 22°C under continuous light (35 µmol m−2 s−1). The plants were transferred to vermiculite in 2-3 weeks and grown at 22°C under a light cycle of 16 h light/8 h dark.
Plasmid construction and transformation using GUS under the KAKU4 promoter control
To construct ProKAKU4:GUS, a genomic fragment encompassing the 2-kb sequence upstream of the coding sequence was cloned into pENTR/D-TOPO (Invitrogen) and then fused upstream of EGFP-GUS in a plant transformation vector (pKGWFS7). The primers used for the amplification of the KAKU4 promoter were the forward primer (5′-CACCGAGGAATACAGGCGAGAACA-3′) and the reverse primer (5′-GGTGAAAGTGAGAGGAGGAG-3′). The sequence motif 5′-CACC-3′ required for cloning into pENTR/D-TOPO was added at the 5′-end of the forward primer. To obtain stable expression, Arabidopsis wild-type plants were transformed with Agrobacterium tumefaciens (GV3101) using the floral dip method (Clough and Bent, 1998).
GUS staining
Floral organs were immersed in 90% acetone, and stored for one or two nights at −20°C. Then, 90% acetone was replaced by 1× phosphate buffer (100 mM NaPO4; 10× stock solution [1M NaPO4] was prepared by mixing 1M Na2HPO4 and 1M NaH2PO4 at a ratio of 61:39; 10× stock solution was diluted 10 times and used as 1× phosphate buffer), which was immediately replaced by the GUS-staining solution (100 mM NaPO4 [pH 7.4], 10 mM EDTA, 0.1% Triton X-100, 1 mM potassium ferricyanide, 1 mM potassium ferrocyanide, 0.5 mg/mL 5-bromo-4-chloro-3-indolyl-β-D-glucuronide cyclohexylammonium). The GUS-staining solution was vacuum infiltrated into the floral organs for 5 min at 20-25°C. The floral organs infiltrated by the GUS-staining solution were incubated for 16 h in the dark at 37°C for GUS-staining reaction. The GUS-staining solution was replaced by 1× phosphate buffer, followed by rinsing with 70% ethanol two times. After rinsing, an ethanol and acetic acid mixture (ethanol: acetic acid = 6:1) was added for specimen preparation. The specimens were mounted with chloral hydrate solution (chloral hydrate: glycerol: water = 8 [g]: 1 [ml]: 2 [ml]) and examined using a differencial interference contrast microscope (DIC) (Axioskop 2 plus system [Carl Zeiss] and a high-sensitivity cooled CCD color camera VB-7010 [Keyence]).
Microscopy
Confocal fluorescence images except for those in Fig. 6 were obtained using a confocal laser scanning microscope (LSM780; Carl Zeiss). The 405 nm line of a blue diode laser, the 488 nm line of a 40 mW Ar/Kr laser, and the 544 nm line of a 1 mW He/Ne laser were used to excite Hoechst 33342, GFP/YFP, and RFP, respectively. Images were acquired with a ×63 1.2 NA water immersion objective (C-Apochromat, 441777-9970-000; Carl Zeiss), a ×40 0.95 NA dry objective (Plan-Apochromat, 440654-9902-000; Carl Zeiss), or a ×20 0.80 NA dry objective (Plan-Apochromat, 440640-9903-000; Carl Zeiss). Data were exported as TIFF files and processed using Adobe Photoshop Elements 9.0 (Adobe Systems) or ImageJ 1.45s (National Institutes of Health; NIH). Fluorescence images presented in Fig. 5A were obtained using an Axioskop2 plus microscope (Carl Zeiss) and a high-sensitivity cooled CCD color camera VB-7010 (Keyence).
Hoechst 33342 staining
Pollen grains and pollen tubes were stained with a solution containing 1 µg/ml Hoechst 33342, 3.7% (w/v) paraformaldehyde, 10% (v/v) dimethyl sulfoxide, 3% (v/v) Nonidet P-40, 50 mM PIPES-KOH (pH 7.0), 1 mM MgSO4, and 5 mM EGTA.
In vitro pollen germination
In vitro pollen germinations were performed as described previously (Boavida and McCormick, 2007). Briefly, pollen grains from full-open flowers were transferred to the medium on a glass slide by gently scraping the surface of the medium by a flower or anthers. Three or four glass slides with the medium containing transferred pollen were placed on Kim Wipes (produced by Nippon Paper Crecia based on partnership with Kimberly Clark) in a square-shaped plastic petri dish (EIKEN CHEMICAL). Kim Wipes wetted with water were put along the vertical wall of the dish to enclose the glass slides before covering the dish with the lid. The petri dish was placed in an incubator with temperature control set to 22°C and continuous light for 3–3.5 h, followed by microscopic observation.
Generation of double nuclear marker line for in vitro pollen tube growth assay
pDM441, a binary vector harboring ProLAT52:NLS-Clover was generated as follows. DNA fragment encoding mClover with A206K was amplified from a template plasmid pPZP221 CloN (Takeuchi and Higashiyama, 2016) by a PCR using a primer set pENTR_NLS_GFP (5’-CAC CAT GGC TCC AAA GAA GAA GAG AAA GGT CAT GGT GAG CAA GGG CGA GGA G −3’) and GFP_R (5’-CTT GTA CAG CTC GTC CAT GCC G −3’). The PCR product was cloned into the pENTR/D-TOPO (Thermofisher) to generate pOR003. LR recombination was performed between the pOR003 with a modified pGWB501 (Nakagawa et al., 2007) harboring LAT52 promoter at the HindIII site (Twell et al., 1990) to produce the pDM441. Agrobacterium GV3101 containing the pDM441 or ProRPS5A:HISTONE 2B-tdTomato (Maruyama et al., 2013) were independently cultured and subsequently used in simultaneous transformation by the floral dip method (Clough and Bent, 1998) to generate double nuclear marker line (ProRPS5A:H2B-tdTomato ProLAT52:NLS-Clover).
Analysis of nuclear size and shape in pollen tubes in the microfluidic device
Pollen grains from the ProRPS5A:H2B-tdTomato ProLAT52:NLS-Clover double nuclear marker line were suspended in an in vitro pollen tube growth medium (Muro et al., 2018) without agarose and placed in a polydimethylsiloxane (PDMS) microfluidic device on a glass slip (Matsunami). The PDMS microfluidic device with 10 µm-width of micro channels was prepared as described previously (Yanagisawa et al., 2017). The device allows a pollen tube to elongate on a focal plane, resulting in easy imaging analysis for hours. After 1h incubation at 22 ºC, fluorescence images of the nuclei labeled with H2B-tdTomato and NLS-Clover in pollen tubes were obtained using a confocal laser scanning microscope SP8 (Leica Microsystems). The images were exported as TIFF files and processed using the Analyze Particles function of ImageJ. A two-tailed homoscedastic Student’s t test was performed using Microsoft Excel. The circularity index was calculated using the equation 4πA/P2 (where A = area of nucleus and P = perimeter of nucleus) and indicates how closely each nucleus corresponds to a spherical shape (a perfect sphere has a circularity index of 1). Any deviation from a circular shape (e.g., elongated, lobulated, or spindle shaped) causes the index to decrease.
Clearing siliques
Fixation and clearing of siliques were performed as described previously (Chen et al., 2015). Silique length was measured using ImageJ software.
Reciprocal crosses
F1 seeds from crosses between wild-type plants and kaku4-3 hetero mutants (kaku4-3/+) were sown on MS medium containing kanamycin sulfate at 70 µg/ml. After 1-2 weeks, the live plants (KanR) and dead plants (KanS) were counted and subjected to chi-squared tests.
Accession numbers
Sequence data from this article can be found in the Arabidopsis Genome Initiative or EMBL/GenBank databases under the following accession numbers: CRWN1 (At1g67230), CRWN2 (At1g13220), CRWN3 (At1g68790), CRWN4 (At5g65770), KAKU4 (At4g31430), Myosin XI-i (At4g33200), Nup136 (At3g10650), SUN1 (At5g04990), SUN2 (At3g10730), WIP1 (At4g26455), WIP2 (At5g56210), WIP3 (At3g13360), WIT1 (At5g11390), and WIT2 (At1g68910).
Results
Remarkably high expression of KAKU4 in mature pollen grains
By analyzing the gene-expression database of A. thaliana (ATTED-II, http://atted.jp/), we found that KAKU4, which encodes a putative nuclear lamina protein, is highly expressed in mature pollen (Fig. 1A) and that the high expression is specific to pollen (Supplemental Fig. S1). Similarly, WIT1, which encodes an outer nuclear membrane protein, is highly expressed in mature pollen (Fig. 1A) but it is also highly expressed in seeds (Supplementary Fig. S1). Expressions of WIT2 and WIP3, which encode other nuclear membrane proteins, do not differ greatly among the tissues (Fig. 1A and Supplementary Fig. S1).
To visualize the activity of the KAKU4 promoter in mature pollen, we generated transgenic plants expressing the β-glucuronidase (GUS) reporter gene under the control of the KAKU4 promoter. Strong GUS signals were detected in pollen grains of the anthers (Fig. 1B, upper panels) and pollen tubes elongating into the stigma (Fig. 1B, lower panels). These results indicate that KAKU4 is predominantly expressed in mature pollen grains.
KAKU4 is highly abundant on the VN envelope and less abundant on the SCN envelope in pollen grains
We examined the subcellular localization of the KAKU4 protein in mature pollen grains with the fluorescent KAKU4 markers. To avoid overexpression of the transgenes, KAKU4-tRFP and KAKU4-EYFP were expressed in the KAKU4-deficient mutant alleles kaku4-2 and kaku4-3, respectively, under the control of the native promoter. Fluorescence signals of KAKU4-tRFP were clearly detected at the VN of each pollen grain (Fig. 2A). At a higher magnification, the fluorescence images showed that KAKU4-tRFP was localized to the nuclear envelope of the VN that were stained with Hoechst 33342 (Fig. 2B, upper panels). With an increased imaging sensitivity, KAKU4-tRFP was also detected on the envelope of two SCNs that were stained with Hoechst 33342 (Fig. 2B, lower panels, arrows). Similar results were obtained with KAKU4-EYFP (see Fig. 3A). The fluorescence levels of the SCNs were much lower than those of the VNs (Fig. 2B and Fig. 3A). Hence, KAKU4 is more densely accumulated in the envelopes of the VNs than in the envelopes of the SCNs.
The dense accumulation of KAKU4 leads to deformation of the VN
The nuclear shapes were compared between the SCN and the VN in the transgenic plants that expressed KAKU4-EYFP in kaku4-3 under the control of the native promoter. The SCNs were nearly spherical in shape and their envelopes were relatively smooth (Fig. 3A). On the other hand, the VN were deeply invaginated (Fig. 3A). The deformation features of the VN are very similar to those of the nuclei in seedlings overexpressing KAKU4-GFP, in which artificial overexpression of KAKU4 causes nuclear envelope deformation in a dose-dependent manner (Goto et al., 2014). This implied that the VN deformation is due to hyperaccumulation of KAKU4 on their envelopes.
Next, the effect of KAKU4 deficiency on the deformation degrees of the VNs was examined with pollen grains from the wild type and the kaku4 mutant alleles. The shapes of the VNs stained with Hoechst 33342 were classified into two types: spherical and non-spherical (Fig. 3B). Non-spherical shapes included elongated shapes and distorted shapes. The proportions of non-spherical shaped VNs were significantly lower in both kaku4-3 and kaku4-4 than in the wild type (Fig. 3C). Taken together, this result indicates that KAKU4 has a role in modulating nuclear shape in pollen.
Involvement of KAKU4 in controlling the migration order of VN and SCNs in elongating pollen tubes
After pollination, a pollen grain germinates and elongates to form a pollen tube, in which the VN and the SCNs migrate towards the ovules (Kawashima and Berger, 2011). Using transgenic plants that expressed KAKU4-tRFP in kaku4-2 under the native promoter and an in-vitro pollen germination assay (Boavida and McCormick, 2007), we were able to observe the KAKU4-labeled VN and the two SCNs migrating towards the tip of elongating pollen tubes (Fig. 4A, left). Like the nuclei in pollen grains, the VN envelopes highly fluoresced, while the SCN envelopes moderately fluoresced (Fig. 4A, right). Similar observations were made with another transgenic plant that expressed KAKU4-EYFP in kaku4-3 under the native promoter (Fig. 4B). In most elongating pollen tubes, the VN preceded the two SCNs (Fig. 4).
To quantitatively analyze the migration order of the VNs and the SCNs, elongating pollen tubes were categorized into three types: (1) the VN-first type, in which the VN preceded the two SCNs (Fig. 5A, upper), (2) the SCN-first type, in which the two SCNs preceded the VN (Fig. 5A, lower), and (3) the together type, in which VN and SCN were close to each other. Most of the wild type pollen tubes (91–93 %) were VN-first type (Fig. 5B). The proportions of VN-first type were markedly reduced to 33–52% in three kaku4 mutant alleles (Fig. 5B), indicating that the control of the migration order of the VN and the SCNs in pollen tubes was impaired in the kaku4 mutant alleles.
We next examined an effect of deficiency of the KAKU4-interacting protein CRWN1 (Dittmer et al., 2007; Sakamoto and Takagi, 2013) on the migration order by using two mutant alleles (crwn1-2 and kaku2) (Goto et al., 2014). The proportions of the VN-first type in these mutant alleles (77-87%) were slightly lower than those of the wild type (Fig. 5B). Taken together, these results suggest that the nuclear lamina candidate proteins KAKU4 and CRWN1 control the migration order of the VN and SCNs in elongating pollen tubes, in which KAKU4 functions more predominantly than CRWN1.
We next compared the nuclear shape and size in the elongating pollen tubes between the wild-type and kaku4-3 plants, by visualizing the VNs and the SCNs with a nuclear marker (H2B-tdTomato) under the constitutive promoter and by visualizing the VNs with another nuclear marker (NLS-Clover) under the VN-specific promoter. The SCNs exhibited moderate difference in the sizes and shapes between the wild type and kaku4-3 (Fig. 6A and Supplemental Fig. S2). On the contrary, compared with the kaku4-3 VNs, the wild-type VNs were larger and highly invaginated (Fig. 6A). To quantitatively evaluate the degree of nuclear deformation, we calculated a circularity index (4πA/P2; where A = area of nucleus and P = perimeter of nucleus). The VN circularity index was much smaller for the wild-type than it was for kaku4-3 in both VN- and SCN-first-type pollen tubes (Fig. 6B, upper). This result indicates that the wild-type VNs are more deformed than the kaku4-3 VNs. In addition, the fluorescence area of the VNs was larger in the wild-type VNs than in kaku4-3 (Fig. 6B, lower). Notably, the kaku4-3 VNs of VN-first-type pollen tubes were significantly smaller than those of the SCN-first-type tubes, although the circularity indices of the VNs of both types of pollen tubes were not significantly different (Fig. 6B). This indicates that having a small size makes it easier for the VNs to precede the sperm cells in elongating pollen tubes.
Slightly decreased seed production and lower pollen competitive ability in kaku4
The preceding observations raised the question of whether the migration order of VN and SCN has an effect on fertilization. To assess the effect of KAKU4 deficiency on fertilization, we analyzed the seed numbers and the silique lengths in the wild type and kaku4 mutant alleles (Fig. 7A). The number of seeds per silique of the kaku4 mutant alleles kaku4-2 and kaku4-3 was moderately decreased to 82% and 87%, respectively, as compared to that number of the wild type (Fig. 7B). The silique was 1 mm shorter in the kaku4 mutants, than in wild-type plants (Fig. 7C). These results indicate that KAKU4 contributed to efficient seed production and proper fruit development.
To test whether the seed reduction in kaku4 was paternally controlled, we reciprocally crossed between wild-type plants and the heterozygous kaku4-3/+ mutant. The KAKU4 mutant gene in kaku4-3 carries a kanamycin resistance gene insert. As shown in Table 1, when kaku4-3/+ was used as pollen donor to the wild type, the number of kanamycin-resistant progeny was significantly smaller than expected, indicating a reduced pollen competitive ability of kaku4. When the wild type was used as the pollen donor to kaku4-3/+, the number of kanamycin-resistant progeny did not significantly differ from the expected values. This suggests that KAKU4 has little or no involvement in functions of pistils in reproduction. Taken together, these results suggest that the VN precedence over SCs in pollen tubes contributes to maintaining proper male transmission efficiency in seed production.
Discussion
In wild-type pollen tubes, the migration order of VN and sperm cells is mostly maintained during their migration, although it occasionally changes (Zhou and Meier, 2014). This implies that the initial positions of the VN and sperm cells when they move from the pollen grain to the pollen tube is important for the VN precedent migration over the two-sperm-cell unit that is surrounded by a membrane (Li et al., 2013; Sprunck et al., 2014; Wudick et al., 2018). In wild-type pollen tubes, the VN enters the pollen tube earlier than sperm cells even though it is expected to be larger than the two sperm cells combined (Mcconchie et al., 1987). In this study, KAKU4 deficiency causes the losses of 1) the irregularity in VN shape and 2) the control of migration order of VN and sperm cells at the same time, suggesting that the VN deformation ability is related to the migration order in the pollen tube. Our observations of wild-type pollen grains showed that VN envelope was deeply invaginated inside the nucleus and that VN shapes were elongated in the pollen tube. These features might allow the VN to easily enter the pollen tube. Hence, we propose that a KAKU4-dependent ability to elongate the VN shapes increases the probability that VN will enter the pollen tube first, resulting in the VN precedent migration over sperm cells in pollen tubes.
Two studies have shown that depolymerization of microtubules can affect the migration order of the VN and sperm cells (Astrom et al., 1995; Heslopharrison et al., 1988). In Galanthus nivalis pollen tubes, microtubule depolymerization with colchicine caused a large increase in the “generative cell lead” (referred to as “SCN first” in this study) (Heslopharrison et al., 1988), and in Nicotiana tabacum pollen tubes, microtubule depolymerization with oryzalin caused a decrease in pollen tubes harboring VN and an increase in pollen tubes harboring generative cells (sperm cells) (Fig. 5 in Astrom et al., 1995). The loss of two proteins essential for the recruitment of γ-tubulin complexes at microtubule organizing centers (GIP1 and GIP2) alters nuclear shape in root tips (Batzenschlager et al., 2013). These results, together with our data, raises the possibility that microtubule depolymerization may cause a change in nuclear shape, which affects the migration order of VN and sperm cells.
While the VN preceded the sperm cells in the majority of wild-type pollen tubes, the sperm cells preceded the VN in the majority of the pollen tubes in multiple mutant alleles of wip, wit, wifi (Zhou and Meier, 2014), and the sun mutant line expressing a dominant-negative gene, in which WIP1 and WIT1 were delocalized from the VN envelope (Lat52pro::ERS-RFP-SUN2Lm sun1-KO sun2-KD) (Zhou et al., 2015). These studies indicate that WIPs and WITs are needed to control the migration order of VN and sperm cells in pollen tubes. VNs were reported to be normal in multiple mutants deficient in WIPs and/or WITs (wip, wit, and wifi) (Zhou and Meier, 2014). However, we cannot exclude the possibility that shape of VN in the mutants’ pollen differs from that in wild-type pollen. The nuclear shape in the leaves and root hairs of the wip mutant allele is similar to that in kaku4 (Zhou et al., 2012). However, because the ratio of “VN first” and “SCN first” is very different between the wip/wit/wifi and kaku4 mutants, the underlying mechanism affecting the migration order of VN and SCNs may be different between WIP/WIT and KAKU4.
Defects in the migration order of VN and sperm cells in wip/wit/wifi (Zhou and Meier, 2014) and kaku4 (this study) caused a decrease in seed reproduction, which suggests that positioning VN first in the pollen tubes is important. In mutants deficient in WIPs and/or WITs, the pollen tubes frequently failed to properly elongate, to burst at the tube tips, and to result in fertilization (Zhou and Meier, 2014). These observations suggest that the proximity of the VN to the growing pollen tube tip is probably critical for pollen tube reception (Zhou and Meier, 2014). The number of seeds in kaku4 mutants was ~80% of that in wild-type plants (Fig. 7B). This result suggests that KAKU4 deficiency does not cause severe defects in pollen tube growth and fertilization. One possible explanation for the impaired competitive ability of pollen in kaku4 is that the elongation speed of SCN-first-type pollen tubes might be slightly lower than that of VN-first-type pollen tubes. Positioning of VN close to the tip may contribute to efficient elongation of pollen tubes.
Supplementary data
The following materials are available in the online version of this article.
Supplementary Fig. S1. Expression of KAKU4, WIT1, WIT2, and WIP3. Transcript levels of KAKU4, WIT1, WIT2 and WIP3 in various tissues. Data were obtained from the resource page of the AtGenExpress project (http://jsp.weigelworld.org/AtGenExpress/resources/) (Schmid et al. 2005). Intensities (absolute values) from select tissues of wild-type plants are shown.
Supplementary Fig. S2. Nuclear shape and size of SCN in pollen tubes of kaku4. Circularity indices and areas of SCNs in the VN-first-type pollen tubes of the wild-type plants and in the VN- and SCN-first-type pollen tubes of kaku4-3 plants. Wild-type pollen tubes showed only VN-first-type positioning. The circularity index is defined as the equation 4πA/P2 (where A = area of nucleus and P = perimeter of nucleus). Means ± standard errors for n = 16 (wild type), 18 (kaku4-3 VN-first), or 16 (kaku4-3 SCN-first). Asterisks indicate a significant difference (Student’s t test, **P < 0.001, *P < 0.005).
Acknowledgements
We are grateful to Tsuyoshi Nakagawa (Shimane University, Japan) for donating the Gateway vectors; and to the Arabidopsis Biological Resource Center for providing the T-DNA tagged lines of A. thaliana. This work was supported by a ‘Specially Promoted Research’ Grant-in-Aid for Scientific Research to I.H-N. (nos. 22000014 and 15H05776), by a Grant-in-Aid for Scientific Research to K.T. (nos. 26711017 and 18K06283), and by a Grant-in-Aid for JSPS Fellows to C.G. (nos. 13J01227 and 17J06391) from the Japan Society for the Promotion of Science (JSPS). This work was also supported by the Human Frontier Science Program to K.T. (RGP0009/2018) from the International Human Frontier Science Program Organization.
Abbreviations
- CRWN
- CROWDED NUCLEI
- SCN
- sperm cell nucleus
- SUN
- Sad1/UNC84 Homology
- VN
- vegetative nucleus
- WIP
- WPP domain–interacting protein
- WIT
- WPP domain–interacting tail-anchored protein.