bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218263; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Synergid calcium ion oscillations define a new feature of pollen tube reception
critical for blocking interspecific hybridization
Nathaniel Ponvert, and Mark A. Johnson*
Department of Molecular Biology, Cell Biology, and Biochemistry; Brown University,
Providence, RI, 02912. [email protected]
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Abstract
Reproductive isolation leads to the evolution of distinct new species, however, the
molecular mechanisms that promote and maintain reproductive barriers are elusive. In
flowering plants sperm cells are immotile and are delivered to female gametes by the
pollen grain which germinates a polarized extension, the pollen tube, into floral tissue.
After growing via polar extension to the female gametophyte, the pollen tube signals with
receptive female cells flanking the egg in two distinct steps. If signaling is successful, the
pollen tube releases sperm cells for fusion with female gametes. To better understand
cell-cell recognition during reproduction, we investigated calcium ion dynamics
associated with interspecific cell signaling between mating partners. We observed that
interspecific pollen tubes successfully complete initial cell-cell signaling, but fail during
later phases of pollen tube reception. Our work refines our understanding of pollen tube
reception as a critical block to interspecific hybridization. Our results also shed light on
the functional significance of the two phases of pollen tube reception, and implicate the
second step as being the stage during which the pollen tube presents its genetic identity.
2 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218263; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Introduction
Pollen tube reception is one of the final potential pre-zygotic barriers to interspecific
hybridization. At high rates during interspecific crosses, sperm are not released and
reproduction is unsuccessful1,2. Loss of function mutations in female gametophyte-
expressed signaling components FERONIA/LORELEI and NORTIA, that participate in
both the first1,3 and second4 stages of pollen tube reception5, respectively, result in pollen
tube reception failure, concurrent with a phenotype similar to what is observed during
interspecific pollination where the pollen tube fails to release sperm. Previous studies
using genetically-encoded calcium ion sensors found that contact between the pollen tube
tip and the female gametophyte initiates calcium ion oscillations in the synergid cells of
the female gametophyte5,23-25 in a FERONIA/LORELEI-dependent manner5. These
oscillations persist for approximately 45 minutes, during the first phase of pollen tube
reception. The second phase of pollen tube reception follows the cessation of the initial
calcium ion oscillations after which a brief rise in cytoplasmic calcium ion concentration
occurs, shortly before pollen tube burst and sperm release5.
Male factors also play roles in pollen tube reception, as loss of pollen tube-expressed
MYB transcription factors results in defective pollen tube reception as well6-8. Given the
similarity in sperm release defects between interspecific crosses and losses of function
in female and male genes, we took a confocal imaging-based approach to uncover how
pollen tube reception plays a role during presentation of species identity. We used a
female gametophyte-expressed genetically-encoded calcium ion sensor to monitor the
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progression of signaling steps during pollen tube reception between interspecific pollen
tubes and feronia and lorelei mutant lines. We also used myb97,101,120 triple-mutant
pollen for crosses with the feronia and lorelei mutants. Strikingly, we found that
interspecific pollen tubes elicit small molecule signaling responses in synergid cells of
other species in a FERONIA/LORELEI-dependent manner, as was observed during
same-species interactions. Our results suggest that genetic identity is communicated
downstream of FERONIA/LORELEI activity. We also observed that myb triple-mutant
pollen tube suitors elicited small molecule responses in female cells despite an ultimate
failure to release sperm. These results are more similar to what we observed during
interspecific pollination than to the lack of oscillations observed during crosses onto lorelei
or feronia null mutants, suggesting that MYB-dependent genes act downstream of
FERONIA/LORELEI and may define a portion of the pollen tube’s genetic identity.
4 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218263; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Results
The species concept includes the idea that members of different species cannot
successfully mate with one another. Interspecific hybridization can fail after a zygote is
produced, however, in many systems pre-zygotic barriers prevent zygote formation1,2,9,10.
Active mechanisms that block pollen tube growth of exotic species have been described
in the Solanaceae, Eucalyptaceae, and Brassicaceae11-14. Additionally, molecular
incongruity resulting from divergence of proteins that mediate gamete or gametophyte
attraction or reception1,2,15 can also result in pre-zygotic barriers to interspecific
hybridization. The molecular recognition events that determine cellular compatibility
between gametes of the same species are only now beginning to be understood15-21. The
final step in flowering plant fertilization, sperm and egg fusion, is not likely to be species-
specific because it is mediated by the highly conserved HAP2 protein, which has been
shown to be functionally interchangeable between distantly related species22. Therefore,
it is not known which male or female signaling components fail during incongruous
interspecific crosses with otherwise compatible mating partners, or even when exactly
during mating partner interactions the communication of genetic identity fails in these
cases when there is no active block to interspecific hybridization. To address these
questions, we used well characterized plant models, including Arabidopsis thaliana, in the
Brassicaceae family.
Same-species pollen tubes induce characteristic synergid cell calcium ion
oscillations
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To begin our analysis of interspecific interactions, we first analyzed interactions between
same-species pollen tubes and synergid cells, as had been done previously5,23-25 (Figure
1, Supplementary Figure 1a, Supplementary Video 1). Although two phases of calcium
oscillations in synergids were identified previously5,23-25, their functional significance
remains unknown. Arabidopsis thaliana lorelei or feronia loss-of-function mutants fail to
receive same-species pollen tubes, resulting in a characteristic pollen tube overgrowth
phenotype similar to that observed during interspecific pollination1-3,15. In lorelei and
feronia mutant females, pollen tube reception defects are accompanied by a severe
attenuation in calcium ion oscillations in the cytoplasm of the synergid cells, indicating
that lorelei and feronia mutant synergid cells are oblivious to the arrival of the pollen tube
tip5 (replicated here in Figure 1c,d,e, Supplementary Figure 1b-c, Supplementary Videos
2,3). To directly measure changes in calcium ion oscillations, we calculated the height of
every peak identified in each calcium ion trace. When we compared the average height
of maxima identified in calcium ion traces from wild-type synergids to the height of maxima
identified in calcium ion traces from lorelei and feronia mutant synergid cells receiving
same species wild-type pollen tubes, we found that in either mutant background there is
a statistically significant reduction in average calcium ion oscillation height, which is
consistent with previously published results5 (Figure 1e).
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Figure 1: Live imaging of calcium ion oscillations during pollen tube reception. a.
Pollen tube growth begins after the pollen grain lands on the stigmatic tissue. b. The
pollen tube makes contact with synergid cells after growing into the embryo sac. Pollen
tube tip denoted with black arrowhead, persistent synergid (relative to pollen tube tip)
marked with yellow arrow, receptive synergid marked with blue arrow. c. The pollen tube
tip participates in a multi-step cell signaling event with the receptive synergid cell and
induces calcium ion oscillations in the cytoplasm of the synergid cells. d. Example traces
(traces with mean peak prominence are presented as examples; all other traces are
presented in the supplement) from crosses onto wildtype, lorelei, and feronia mutant
females using wildtype Arabidopsis thaliana pollen. Calcium ion trace from left synergid
cell plotted in yellow and right synergid cell plotted in blue. e. Plot of calcium transient
prominence observed during crosses onto female genotypes (wildtype n=10 traces n=128
7 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218263; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
peaks, lorelei n=10 traces n=161 peaks, feronia n=10 traces n=118 peaks). Statistical
significance calculated using analysis of variance (ANOVA, α = 0.05).
8 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218263; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Interspecific pollen tubes induce calcium ion oscillations in synergid cells of
different species
We next asked whether calcium ion oscillations occur in synergid cells during interspecific
crosses using Arabidopsis thaliana females receiving pollen tubes from Arabidopsis lyrata
or Olimarabidopsis pumilia and if the nature of the oscillation is tied to specific recognition.
We chose A. lyrata and O. pumilia as pollen donors, as they are predicted to be diverged
from A. thaliana ~4-10 million years ago (MYA) and ~10-14 MYA, respectively, thereby
serving as representatives of closely- and more distantly-related species26-28.
When we performed crosses using pollen from either Arabidopsis lyrata (n = 10) or
Olimarabidopsis pumilia (n = 10), we observed the initiation of calcium ion oscillations in
wild-type Arabidopsis thaliana synergid cells (Figure 2a,c, Supplementary Figure 2a,
Supplementary Figure 3a, Supplementary Videos 4,7). However, the calcium ion
oscillations induced by either type of interspecific pollen tube (Figure 2) did not appear to
follow the stereotypical pattern of progression observed during same-species crosses
described above (Figure 1)5,23-25. During interspecific interactions, pollen tubes were able
to induce calcium ion oscillations in synergid cells, similarly to what is observed during
phase one of same-species pollen tube reception, but we very rarely observed a
progression into the second phase of reception (a shift from calcium ion oscillation to
constitutive increase in calcium ion concentration, termed ‘plateau’) and we did not
observe pollen tube burst in any interspecific cross, indicating that induction of calcium
ion oscillations alone is not sufficient to induce pollen tube burst. These results suggest
9 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218263; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
that A. lyrata and O. pumilia pollen tubes are able to successfully signal their arrival to a
gametophyte of a different species, but that successful sperm release requires congruity
in a distinct molecular recognition event that follows initiation of calcium oscillations.
Calcium ion oscillations induced by interspecific pollen tubes require the
FERONIA/LORELEI receptor complex
To investigate whether the calcium ion oscillations observed during interspecific crosses
are FERONIA/LORELEI signaling complex-dependent, we crossed A. lyrata or O. pumilia
pollen onto A. thaliana lorelei or feronia females. If the calcium ion oscillations observed
during interspecific pollinations are also dependent on FERONIA/LORELEI as they are
during same-species pollination, then an attenuation of calcium ion responses as seen in
same-species pollination events would be expected5 (Figure 1d,e). Indeed, we observed
an attenuation of calcium ion oscillations in lorelei and feronia mutant females receiving
interspecific pollen tubes (Figure 2a-d, Supplementary Figure 2b-c, Supplementary
Figure 3b-c, Supplementary Videos 5,6,8,9), similar to those previously reported for
lorelei and feronia mutants receiving wildtype A. thaliana pollen tubes5 (replicated in
Figure 1, Supplementary Figure 1b-c, Supplementary Videos 2,3). These results
suggested that FERONIA and LORELEI are required for the interspecific pollen tube-
induced calcium ion oscillations but the failure to present congruous identification signals
to the receptive synergid cell occurs downstream of FERONIA/LORELEI activation, and
a failure of this downstream step results in failure to release sperm.
10 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218263; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Figure 2. Interspecific pollen tubes elicit calcium ion oscillations in wildtype
synergid cells. a. Example traces from crosses onto wildtype, lorelei, and feronia mutant
females using wildtype Arabidopsis lyrata pollen. b. Plot of calcium transient prominence
observed during crosses onto female genotypes (wildtype n=10 traces n=82 peaks, lorelei
n=10 traces n=96 peaks, feronia n=10 traces n=121 peaks). Statistical significance
calculated using analysis of variance (ANOVA, α = 0.05). c: Example traces from crosses
onto wildtype, lorelei, and feronia mutant females using wildtype Olimarabidopsis pumilia
pollen. Calcium ion trace from left synergid cell plotted in yellow and right synergid cell
plotted in blue. d. Plot of calcium transient prominence observed during crosses onto
11 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218263; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
female genotypes (wildtype n=10 traces n=90 peaks, lorelei n=10 traces n=121 peaks,
feronia n=10 traces n=81 peaks). Statistical significance calculated using analysis of
variance (ANOVA, α = 0.05).
12 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218263; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
myb97,101,120 transcription factor triple-mutant male pollen tubes phenocopy
interspecific signaling defects
We next explored the possibility that the pollen tube is the source of signaling molecules
that underlie the molecular recognition of the pollen tube by the synergid cell. To date,
the only male-specific mutations resulting in failure of sperm release are triple null
mutants in the pollen tube-expressed MYB97, 101, and 120 transcription factors2,6-8,29.
The myb97,101,120 triple mutant displays a characteristic pollen tube overgrowth
phenotype after making contact with the female gametophyte that is similar to the
phenotype observed with feronia and lorelei mutant females, and to what is observed
during interspecific crosses1-3,6-8,15,29. However, it is not known when MYB-dependent
genes act during reception, and/or if they interact with FERONIA/LORELEI signaling
complex. Given the similarity in pollen tube overgrowth phenotypes observed during
interspecific pollinations and myb97,101,120 mutant pollinations, we hypothesized that
the MYB-dependent gene regulatory network could constitute some portion of the genetic
identity that must be properly perceived by the female gametophyte during pollen tube
reception.
To more precisely define the roles of male factors in pollen tube reception, we used
myb97,101,120 triple mutants as pollen donors in same-species crosses. When
myb97,101,120 pollen was crossed to wild-type A. thaliana pistils, pollen tubes elicited
aberrant calcium ion oscillations within wild-type synergid cells (Figure 3a, Supplementary
Figure 3a, Supplementary Video 10). These results supported the possibility that
13 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218263; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
myb97,101,120 pollen tubes only fail during the second phase of pollen tube reception,
similar to interspecific pollinations, and that the MYB-dependent genes could define some
portion of the pollen tube’s genetic identity that is presented to the female gametophyte.
Calcium ion oscillations induced by myb mutant tubes require FERONIA/LORELEI
Induction of calcium ion oscillations by myb97, 101, 120 pollen tubes in wild-type synergid
cells suggested that the role of MYB factors in pollen tube reception is downstream of
FERONIA/LORELEI. To test this possibility, we performed same species crosses
between myb triple mutant pollen on lorelei and feronia mutant females. We observed
that, as in crosses with wild-type and interspecific pollen tubes, calcium ion oscillations
were severely attenuated in lorelei and feronia mutant females receiving myb97,101,120
mutant pollen tubes (Figure 3a,b, Supplementary Figure 3b-c, Supplementary Videos
11,12). In the majority of crosses (8/10 in lorelei x myb and 6/10 in feronia x myb) calcium
transients are largely ablated, with the remaining fraction displaying an occasional
calcium ion transient. In such cases, calcium ion transients persist briefly and are, on
average, less prominent than in crosses onto wildtype females (Figure 3b). These results
supported the conclusion that the MYB gene regulatory network plays a role downstream
of FERONIA/LORELEI, during the second phase of pollen tube reception when mate
genetic identity is vetted by the female gametophyte.
14 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218263; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Figure 3. myb 97, 101, 120 pollen tubes elicit calcium ion oscillations lacking a
second phase in wildtype synergid cells. a. Example traces from crosses onto
wildtype, lorelei, and feronia mutant females using myb97,101,120 triple-mutant
Arabidopsis thaliana pollen. Calcium ion trace from left synergid cell plotted in yellow and
right synergid cell plotted in blue. b. Plot of calcium transient prominence observed during
crosses onto female genotypes (wildtype n=10 traces n=115 peaks, lorelei n=10 traces
n=137 peaks, feronia n=10 traces n=103 peaks). Statistical significance calculated using
analysis of variance (ANOVA, α = 0.05).
15 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218263; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Calcium ion oscillation frequency is not the sole determining factor of pollen tube
burst success rate
Next, we asked whether the calcium ion oscillation signature recorded from wild-type
Arabidopsis thaliana synergid cells responding to same-species, interspecific, and myb
mutant pollen tubes differs. Previous studies have found that cell-cell signaling between
roots and fungal or bacterial symbionts initiates calcium ion oscillations in plant root cells,
and propose that the frequency of the oscillations depends on the species identity of the
symbiont30-32. When we measured the frequency of the calcium ion oscillations induced
by pollen tubes in our system, we found that Arabidopsis lyrata and Olimarabidopsis
pumilia tubes induce oscillations with similar frequency to those induced by same-species
pollen tubes. However, the calcium ion oscillations induced by myb-mutant pollen tubes
occur with a lower frequency than those induced by wild-type Arabidopsis thaliana pollen
tubes (Figure 4, Supplementary Figure 5). This suggests that pollen tubes lacing the
MYB97,101,120 gene regulatory network are defective in communicating with synergid
cells during the second phase of pollen tube reception. However, we concluded that while
the frequency of the calcium ion oscillations induced by interspecific and mutant pollen
tubes is variable, we did not observe a connection between the frequency of the calcium
ion oscillations and interspecific identity.
16 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218263; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Figure 4. Calcium ion oscillation frequency is lower in synergid cells interacting
with myb mutant pollen tubes, but not in synergid cells interacting with
interspecific pollen tubes. a. Frequency of calcium ion oscillations was calculated for
wild-type Arabidopsis thaliana synergid cells responding to wild-type same-species pollen
tubes, myb triple-mutant same-species pollen tubes, Arabidopsis lyrata, and
Olimarabidopsis pumilia pollen tubes was calculated by dividing left- and right-synergid
ROIs and applying thresholding based on oscillation prominence. Oscillations above the
threshold of 5x the minimum prominence observed in the trace are marked with ‘+’. The
median replicate is shown. b. myb triple-mutant pollen tubes induce calcium ion
oscillations that occur with lower frequency than wild-type same-species induced
oscillations, but interspecific pollen tubes induced oscillations with a frequency not
significantly different from same-species pollen tubes. Statistical significance calculated
using analysis of variance (ANOVA, α = 0.05).
17 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218263; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Discussion
Here we report on calcium ion oscillations that are induced in the cytoplasm of synergid
cells after the arrival of pollen tubes of either an exotic species (A. lyrata or O. pumila) or
same-species (wild type or myb97,101,120 triple mutant). We found that in both cases
the initiation of calcium ion oscillations was dependent on the FERONIA/LORELEI
signaling complex. During crosses with the myb mutant, calcium ion oscillations occurred
with a lower frequency than during wild-type crosses, but interspecific pollen tubes had
no significant effect on calcium ion oscillation frequency, suggesting that the frequency of
calcium ion oscillations may not be indicative of genetic identity in this system. Our results
suggest that pollen tube reception defects (overgrowth and failure to release sperm
manifested as a pollen tube coiling phenotype) during interspecific crosses, and during
crosses using myb97,101,120 mutant pollen is not due to a failure in FERONIA/LORELEI
function, but rather that after FERONIA and LORELEI recognize the arrival of the pollen
tube tip, there is a defect in pollen tube reception that occurs during the second phase.
Our findings suggest that the function of the second phase of pollen tube reception is to
scrutinize the genetic identity of the pollen tube and present a barrier to interspecific pollen
tubes. Additionally, our findings indicate that the MYB97, 101, 120 gene regulatory
network may define some portion of the molecular species identity that is presented to
the female gametophyte. Finally, our results suggest that the factor or factors that the
FERONIA/LORELEI signaling complex is recognizing may be independent from MYB
regulation.
18 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218263; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
It is unclear at this point what factors the FERONIA/LORELEI signaling complex is
responding to in the case of pollen tube reception. In sporophytic tissue it is known that
FERONIA is capable of binding and responding to Rapid Alkalinization Factor (RALF)
peptides33-35. It has also been established that pollen tube expressed paralogs of
FERONIA interact with pollen tube- and ovule-expressed RALFs that control pollen tube
cell wall integrity (pollen tube burst and sperm release) in conjunction with pollen tube
expressed-LORELEI paralogs36-38. However, no pollen tube-expressed RALF has been
identified that interacts with FERONIA/LORELEI directly. It may be the case that physical
deformation of the synergid cell surface by the growing pollen tube tip is sufficient to
initiate the FERONIA signaling cascade as it is in seedlings39, or that pollen-expressed
ligands (e.g. RALF peptides) are unaffected by myb mutations.
Previous reports1 have suggested that FERONIA may be involved in scrutinizing the
pollen tube’s genetic identity, but our results suggest that species recognition occurs
downstream of the activation of this receptor complex. Using the calcium ion oscillation
signature as a readout for points of failure during pollen tube reception has allowed us to
order the signaling components and genetic identity factors in this signaling pathway. In
plants, signaling between the pollen tube and cells of the pistil and female gametophyte
provide an opportunity for investigating molecular interactions determining interspecific
mating success. Pollen tube reception (sperm release) breaks down in crosses between
closely related species and we have shown that pollen tube-induced and
FERONIA/LORELEI-dependent calcium ion oscillations occur in interspecific crosses but
19 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218263; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
are insufficient to induce sperm release. Future work will be aimed at determining the
factors that function downstream of FERONIA/LORELEI to decode species identity.
20 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218263; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Data availability statement
Raw data for the following figures is provided as listed. All original confocal time-lapse
datasets and region of interest coordinates are available: https://doi.org/10.26300/b6za-
nt53.
Figure 1-3 - Raw trace data provided in Supplementary File 1
Figure 1-3 – Raw statistical data provided in Supplementary File 2
Code availability statement
All code used in this manuscript is supplied as supplementary data. Data for this study
was collected using Zen Black (available through Zeiss), and the opensource ImageJ
platform (available at https://imagej.nih.gov/ij). Ratiometric calculations were performed
using a custom ImageJ .IJM file (Supplementary File 3) written by NDP that depends on
the RatioPlus plugin (available at https://imagej.nih.gov/ij/plugins/ratio-plus.html). Data
were analyzed using MATLAB (available at
https://www.mathworks.com/products/matlab.html). The MATLAB script used to organize
fluorescent data for plotting was written by NDP and is supplied as Supplementary File 4.
The MATLAB script used to plot concatenated data as colored lines was written by NDP
and is provided as Supplementary File 5. The MATLAB script used to plot concatenated
data as boxplots was written by NDP and is supplied as Supplementary File 6.
Supplementary File 6 relies on the ViolinPlot repository (available at
https://github.com/bastibe/Violinplot-Matlab).
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Author contributions
NDP and MAJ designed and planned the experiments. NDP performed the experiments
and collected the data. NDP analyzed the data. NDP and MAJ wrote the manuscript.
Acknowledgements
We thank Ravishankar Palanivelu, Sharon Kessler, Jeff Harper, and Alexander Leydon
for generating and/or providing transgenic lines for this study. We thank the Leduc
Imaging Core Facility at Brown University and Geoff Williams for providing resources and
expertise regarding time-lapse imaging. We thank Alison DeLong, Judith Bender, and
Jenna Kotak for helpful discussions regarding this study. We thank Sherry Warner for
maintaining plantings for this study. Funding for this project was provided by US National
Science Foundation grant IOS-1353798 (MJ) and US National Institutes of Health
Training Grant #T32-GM007601.
Competing interests
The authors declare no competing interests
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Supplementary Figures
Supplementary Figure 1. a. 10 replicate time-lapse calcium ion traces of
pMYB98:YC3.60 in Wildtype Arabidopsis thaliana crossed as female with Wildtype
Arabidopsis thaliana pollen. b. 10 replicate time-lapse calcium ion traces of
pMYB98:YC3.60 in lorelei-mutant background Arabidopsis thaliana crossed as female
with Wildtype Arabidopsis thaliana pollen. c. 10 replicate time-lapse calcium ion traces of
pMYB98:YC3.60 in feronia-mutant background Arabidopsis thaliana crossed as female
with Wildtype Arabidopsis thaliana pollen. For each trace left synergid region of interest
plotted with blue line and right synergid region of interest plotted with yellow line. Traces
shown in Figure 1 highlighted with red dot at left.
23 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218263; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Supplementary Figure 2. a. 10 replicate time-lapse calcium ion traces of
pMYB98:YC3.60 in Wildtype Arabidopsis thaliana crossed as female with Wildtype
Arabidopsis lyrata pollen. b. 10 replicate time-lapse calcium ion traces of
pMYB98:YC3.60 in lorelei-mutant background Arabidopsis thaliana crossed as female
with Wildtype Arabidopsis lyrata pollen. c. 10 replicate time-lapse calcium ion traces of
pMYB98:YC3.60 in lorelei-mutant background Arabidopsis thaliana crossed as female
with Wildtype Arabidopsis lyrata pollen. For each trace left synergid region of interest
plotted with blue line and right synergid region of interest plotted with yellow line. Traces
shown in Figure 2 highlighted with red dot at left.
24 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218263; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Supplementary Figure 3. a. 10 replicate time-lapse calcium ion traces of
pMYB98:YC3.60 in Wildtype Arabidopsis thaliana crossed as female with Wildtype
Olimarabidopsis pumilia pollen. b. 10 replicate time-lapse calcium ion traces of
pMYB98:YC3.60 in lorelei-mutant background Arabidopsis thaliana crossed as female
with Wildtype Olimarabidopsis pumilia pollen. c. 10 replicate time-lapse calcium ion traces
of pMYB98:YC3.60 in feronia-mutant background Arabidopsis thaliana crossed as female
with Wildtype Olimarabidopsis pumilia pollen. For each trace left synergid region of
interest plotted with blue line and right synergid region of interest plotted with yellow line.
Traces shown in Figure 2 highlighted with red dot at left.
25 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218263; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Supplementary Figure 4. a. 10 replicate time-lapse calcium ion traces of
pMYB98:YC3.60 in Wildtype Arabidopsis thaliana crossed as female with myb97,101,120
triple-mutant Arabidopsis thaliana pollen. b. 10 replicate time-lapse calcium ion traces of
pMYB98:YC3.60 in lorelei-mutant background Arabidopsis thaliana crossed as female
with myb97,101,120 triple-mutant Arabidopsis thaliana pollen. c. 10 replicate time-lapse
calcium ion traces of pMYB98:YC3.60 in feronia-mutant background Arabidopsis thaliana
crossed as female with myb97,101,120 triple-mutant Arabidopsis thaliana pollen. For
each trace left synergid region of interest plotted with blue line and right synergid region
of interest plotted with yellow line. Traces shown in Figure 3 highlighted with red dot at
left.
26 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218263; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Supplementary Figure 5. a. 10 replicate time-lapse calcium ion calcium ion traces of
pMYB98:YC3.60 in Wildtype Arabidopsis thaliana crossed as female with Wildtype
Arabidopsis thaliana pollen. b. 10 replicate time-lapse calcium ion traces of
pMYB98:YC3.60 in Wildtype Arabidopsis thaliana crossed as female with myb97,101,120
triple-mutant Arabidopsis thaliana pollen. c. 10 replicate time-lapse calcium ion traces of
pMYB98:YC3.60 in Wildtype Arabidopsis thaliana crossed as female with Wildtype
Arabidopsis lyrata pollen. For each trace left synergid region of interest plotted with blue
line and right synergid region of interest plotted with yellow line. d. 10 replicate time-lapse
calcium ion traces of pMYB98:YC3.60 in Wildtype Arabidopsis thaliana crossed as female
with Wildtype Olimarabidopsis pumilia pollen. For each trace calcium ion peaks that were
used for frequency analysis (with a prominence greater than 5x minimum prominence
observed) are marked with ’+’ for left synergid ROI and ’x’ for right synergid ROI).
27 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218263; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Materials and Methods
Transgenic Lines
Wildtype and lorelei-mutant background pMYB98:YC3.6040 lines were supplied by Dr.
Ravishankar Palanivelu’s Laboratory at the University of Arizona. pMYB98:YC3.60 was
introgressed into the nta-1 mutant background and the fer-4 mutant background which
were gifted by Dr. Sharon Kessler’s Laboratory at Purdue University. The Arabidopsis
thaliana pLAT52:dsRed line was gifted by Dr. Jeffery Harper’s Laboratory at The
University of Nevada, Reno. Olimarabidopsis pumilia was transformed with
pLAT52:dsRed using Agrobacterium-mediated transformation by Dr. Alexander Leydon
(Current address: Department of Biology; University of Washington, Seattle). The myb97,
101, 120 was generated by Dr. Alexander Leydon. All genotyping primer sequences used
for this study are available upon request.
Arabidopsis growth, pistil dissection, and time-lapse microscopy
Arabidopsis plants were grown under 16-hours-light, 8-hours-dark until mature. Pistils
were emasculated 24 hours before dissections. Pistils were pollinated and left for 45
minutes before dissection. 200 microliters of Arabidopsis pollen growth media (5mM KCl,
0.01% H3BO3, 5mM CaCl2, 1mM MgSO4, 10% Sucrose, 1.5% NuSeive GTG Agarose,
pH 7.6) was allowed to cool as a pad in a MatTek 35mm glass bottom dish with No. 1.5
coverglass (part no. P35G-1.5-20-C, https://www.mattek.com/store/p35g-1-5-20-c-
28 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218263; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
case/). After media had cooled, pistils were dissected onto media and left in a humidity
chamber at 22°CC for 5 hours. Dishes were imaged on a Zeiss LSM Laser Scanning
Confocal Microscope at 20x optical zoom with 3.5x digital zoom. Images were taken with
a 10 second interval starting before pollen tube contact with synergid cells and continuing
for 1 hour after contact. For Arabidopsis lyrata, because we do not have a pollen-
expressed fluorescent reporter in this species, we could not track the growth of the pollen
tube tip after it had grown through the micropyle. To address this, during wildtype A.
thaliana pMYB98:YC3.60 x A. lyrata crosses, we used the initial calcium ion transient
observed in synergid cells as the marker for contact between the pollen tube tip and
synergid cell. During lorelei pMYB98:YC3.60 x A. lyrata crossesand feronia
pMYB98:YC3.60 x A. lyrata crosses, we used deformation of the synergid cell as the
marker for contact between the pollen tube tip and synergid cell, as in these mutant
backgrounds calcium ion oscillations are largely abolished4. For crosses using A. thaliana
or O. pumilia pollen donors, pollen-expressed red fluorescence allowed us to track the
pollen tube as it grew into the embryo sac and first contact was defined visually.
Time-lapse data set analysis
To analyze time-lapse datasets, we use a custom ImageJ (https://imagej.nih.gov/ij/)
macro (supplied in Supplementary File 4 as a commented, editable .ijm file). Any time-
lapse movies suffering from drift were corrected using the MultiStackReg plugin
(http://bradbusse.net/sciencedownloads.html). Supplementary File 4 splits multi-channel
time-lapse datasets into constituent channels, asks the user to draw regions of interest
29 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218263; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
around the left and right synergid cells, subtracts any pollen-expressed fluorescence from
synergid cell channels, and uses the RatioPlus plugin
(https://imagej.nih.gov/ij/plugins/ratio-plus.html) to calculate relative FRET efficiency
within the regions of interest. Following this, we remove bright outliers and despeckle the
output to remove noise resulting from our data collection process. The ratiometric results
are saved automatically as .csv files, along with the summed projection over which the
user draws the regions of interest around the synergid cells, the coordinates of regions of
interest themselves, and a flattened composite time-lapse to the directory from which the
time-lapse dataset is opened.
FRET data analysis
Data was analyzed using MATLAB (available at
https://www.mathworks.com/products/matlab.html). The MATLAB script used to
concatenate fluorescent data (Supplementary File 1) was written by NDP and is supplied
as Supplementary File 5. The MATLAB script used to plot concatenated data as colored
lines was written by NDP and is provided as Supplementary File 6. The MATLAB script
used to plot concatenated data as boxplots was written by NDP and is supplied as
Supplementary File 7. Supplementary File 7 relies on the ViolinPlot repository (available
at https://github.com/bastibe/Violinplot-Matlab). For Figures 1-3, calcium ion peaks were
analyzed if they were greater than 1.1x the minimum prominence value registered for
their respective trace. This cutoff value was chosen such that noise could be excluded
from the analysis while still allowing traces with few/small prominences (those onto lorelei
30 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218263; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
or feronia females) could still be analyzed. For Figure 4, calcium ion peaks with
prominences greater than 5x the minimum prominence value registered for their
respective traces were analyzed.
Data plotting and figure construction
For each cross scheme all ten replicates were sorted according to average calcium peak
prominence. Traces shown in Figures 1-3 were chosen as they are the trace with median
peak prominence. For each statistical plots in Figures 1-4 median value denoted by red
line. 95% confidence interval about the median is denoted by notches. Box defines
interquartile range. Length of whiskers is calculated as 1.5*IQR. ANOVA and post-hoc
multiple comparison of means supplied as Supplementary File 2 and Supplementary File
3.
31 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.23.218263; this version posted July 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
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