1

Extended Summary

Deciphering the role of antherozoid specific DNA methyltransferases in

Physcomitrella patens

Sónia Alexandra Gomes Pereira

Supervisors: Doctor Leonilde de Fátima Morais Moreira and Doctor Jörg-Dieter Becker

Abstract

Cytosine methylation represents the most common DNA modification in eukaryotic genomes. In

plants, 5-mC is found in CG, CHG and CHH contexts, being catalyzed by DNA methyltransferases. In

antherozoids of Physcomitrella patens expression of de novo DNA methyltransferases is limited to

PpDRM2 and PpDNMT3b, standing in stark contrast with the broad expression of other DNA

methyltransferases during the life cycle of this model bryophyte. Given the importance of DNA

methylation for genome integrity, this observation prompted us to study the role of de novo methylation

during sexual reproduction in Physcomitrella.

We obtained two independent Δdrm2 knockout lines and analyzed their fertilization rates in

comparison to the wild -type. In the F0 lower rates were detected for Δdrm2#1, but not for Δdrm2#2. F1

and F2 lines showed no variation in rates, indicating a possible compensatory mechanism after the first

fertilization. Based on an observation that prolonged cold storage of spores led to irregular shaped

colonies with gametophores in wild-type and smaller and round colonies lacking gametophores in

Δdrm2, we performed a time-course phenotyping experiment. To this end, methods for high-throughput

colony area and dry weight assessment were established. The variation in colony growth was observed

again, but it could not be linked to prolonged cold storage of spores, nor to similar phenotypes reported

for a number of other mutants.

Furthermore, we developed a novel protocol for time-efficient isolation of Physcomitrella

antherozoids based on FACS of fluorescein diacetate labelled antherozoids, a method crucial for future

studies of their methylation profiles.

Key-words: Physcomitrella patens; DNA methylation; de novo methyltransferases; DRM2

knockout; antherozoids; Fluorescence-activated cell sorting.

Introduction

Cytosine methylation (5-mC) is present in most eukaryotic genomes being a stable epigenetic

modification catalyzed by DNA methyltransferases (DMTases). In animals, from 3 to 8% of all cytosines

are methylated and almost exclusively in the CG context, although some asymmetric methylated

cytosines (CHH context, H = A, T or C) have also been detected (Ichiyanagi et al., 2013). In plant

genomes, from 6 to 30% of all the cytosine nucleotides are methylated and these are present in all three

sequence contexts: CG, CHG (symmetrical methylations) or CHH (asymmetrical methylation) (Malik et

al., 2012; Noy-Malka et al., 2014). In plants, the DMTases are categorized into four subfamilies: DNMT2,

METs, CMTs and domains rearranged methyltransferase (DRMs) (Malik et al., 2012). In animals, MET

proteins are known homologs of the DNMT1 proteins and DRM proteins are replaced by the de novo

methyltransferases group constituted by DNMT3 proteins (Kuhlmann et al., 2014).

CHH methylation is maintained by the persistent activity of de novo methyltransferases capable

of methylating previously unmethylated DNA either by the RNA-directed DNA methylation (RdDM)

pathway that requires DRMs action, or independently of this process by CMT2’s activity. The first

evidences of DNMT3a and DNMT3b activity as DNA methyltransferases in vivo comes from a study

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conducted in 1999 by Hsieh. Okano et al. (1999) found that DNMT3a and DNMT3b are essential for de

genome-wide de novo methylation and for mammalian development.

The model moss Physcomitrella patens (P. patens) is the first bryophyte to have its genome

sequenced (~480Mbp). Its life cycle starts with the germination of a haploid spore into branched

filamentous protonema tissue composed of both chloronema and caulonema cells (Strotbek et al.,

2013). The transition to the adult gametophore begins by the development of gametophores with a leaf-

like shape and rhizoids at their base. Sexual reproduction is initiated by the development of both female

archegonia and male antheridia, gamete producing organs at the tip of the gametophore. Physcomitrella

patens antherozoids (male gametes) are strikingly similar to the sperms of certain animals in the way

that they are flagellated cells and swim. Upon fertilization, the diploid sporophyte starts to develop. Inside

the sporophyte capsule, meiosis will take place and about 4000 to 6000 haploid spores are produced.

These spores will mature inside the sporophyte and afterwards the capsule breaks open releasing the

spores for propagation (Strotbek et al., 2013).

P. paten’s main advantage is its high frequency of homologous recombination, that allows an

efficient gene targeting and the generation of stable mutant lines to conduct reverse genetics

experiments (Prigge and Bezanilla, 2010; Schaefer and Zrÿd, 2001; Strotbek et al., 2013). P. patens

allows the study of gene function, the decoding of developmental mechanisms present in ancestral land

plants and the colonization of terrestrial environments by plants (Prigge and Bezanilla, 2010).

In P. patens, all three contexts of cytosine methylation were found to be enriched in repetitive

regions, reduced on gene bodies and almost absent around the transcriptional start site. The levels of

5-mC residues present on P. patens whole plants nuclear genome were found to be around 29.5% of

CG, 29.7% of CHG and 23.2% of CHH methylated cytosines. P. patens levels of CHH methylation

(~23%) were the highest of the 17 eukaryotic genomes analysed by Zemach et al., 2010.

In a study published in 2012, Malik et al. used zebularine (a DMTase inhibitor) to study the effects

of a genome-wide loss of 5-mC on P. patens physiology and development. The authors detected

developmental defects and concluded that DNA methylation levels have a profound effect on growth

and differentiation of cells during gametophyte development in P. patens. In the same study, the authors

used an in silico approach to detect genes encoding DMTases in the P. patens genome, revealing the

presence of 7 loci possibly encoding such enzymes. Five of those genes appear to code for

methyltransferases homologous to the ones present in flowering plants (MET1, CMT, DNMT2, DRM1

and DRM2), while two others appear to be related to the human DNMT3a and DNMT3b

methyltransferases (Malik et al., 2012).

P. patens is the earliest diverged plant in which a CMT gene was identifed, and knock-out (K.O.)

mutations of this gene cause developmental deffects such as arrest of protonema growth and absence

of gametangia and sporophytes. Overal, CMT mutants revealed genome-wide hypomethylation with an

almost complete loss of CHG methylation, while no significant changes in CG methylation were

observed (Dangwal et al., 2014; Noy-Malka et al., 2014). In 2015, Yaari and co-workers studied the

function of P. paten’s MET1, showing a dramatic loss of CG methylation and a reduction of CHG

methylation only at CCG sites when the MET1 gene was disrupted. Physiologically P. patens met1

mutant plants develop normally but they fail to form sporophytes, indicating that MET1 may have an

essential role in either gamete formation, fertilization or sporophyte development. As for CMT mutants,

MET1 mutant microarray results also detected upregulation of a subset of repetitive sequences,

suggesting that both enzymes can cooperate to silence these repetitive sequences (Yaari et al., 2015).

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Table 1: Presence and absence call for the expression of Physcomitrella patens’ DNA methyltransferase genes. Gene identification from version 1.6 of the genome, the dots represent expression detected and the absence of dots means that no significant expression level was detected. Red dots represent maintenance DMTase gene MET1; Purple dots represent CMT gene; Blue dots represent de novo DMTase genes with homology to A. thaliana’s: DRM1 (dark blue) and DRM2 (lighter blue); Green dots represent de novo DMTases genes with homology to the human ones: DNMT3a (dark green) and DNMT3b (lighter green) (Adapted from Hernández-Coronado, 2015).

Recently, microarray analysis of the different tissues of P. patens, covering both the vegetative

stages as well as the reproductive phases of development were used to generate a transcriptome atlas

of this plant (Hernández-Coronado, 2015; Ortiz-Ramírez et al.). Using this transcriptome atlas the

expression of the different DMTase genes in P. patens’ genome was explored (Table 1). DRM1 and

DRM2 expression patterns seem to complement each other, since DRM1 is expressed in all the tissues

except the antherozoids and DRM2 transcripts are only detected in these gametes. DNMT3a seems to

have the same expression profile as DRM1, while DNMT3b transcripts are detected in the antherozoids

and the S3 stage of sporophyte development (Table 1, Hernández-Coronado, 2015; Ortiz-Ramírez et

al.).

The data used for the transcriptome atlas involved samples from P. patens’ antherozoids. These

cells are released in clusters of approximately 50 to 150 cells, and in order to collect enough

antherozoids for transcriptomic analysis about 200-400 clusters were required. The method used for the

collection of such samples was based on the manual dissection of mature antheridia into water, until the

antherozoid clusters were released. The collection of each individual cluster was achieved under an

inverted microscope with a micromanipulator (Hernández-Coronado, 2015).

We aimed to better understand the epigenetic mechanisms acting during plant reproduction using

P. patens as our model organism, focusing on the antherozoid, which contribute directly to the next

generation. In this work we characterized two independent K.O. mutant lines for DRM2 (Δdrm2#1 and

Δdrm2#2), previously generated in our laboratory, namely their fertilization rate and colony growth. We

also attempted to generate Δdnmt3b as well as to develop a time-efficient method to collect P. patens

antherozoids by fluorescence-activated cell sorting (FACS).

Materials and Methods

Physcomitrella patens maintenance and growth: Physcomitrella patens lines used in this work

were maintained by vegetative propagation, grown in solid KNOPS+T media for 6 to 7 days. Tissue

grew at 25ºC with 16 hours on light and 8 hours on dark daily cycles. To allow full development of the

plants, tissue was transferred to jiffies. Jiffies were kept under the same conditions as the tissue plates

during three weeks, after which the reproductive stage of P. patens life cycle was induced. This was

achieved by changing the conditions to 17°C under short-day conditions comprising 8 hours of light and

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16 hours of dark cycles. The plants were maintained in these conditions until they completed their life

cycle (8 weeks after the induction of sexual reproduction) (Cove, 2005; Strotbek et al., 2013). Jiffies

were watered when required to keep a high level of humidity.

Fertilization rates assessment: fertilization rate was assessed using plants grown in jiffies 6 weeks

after induction of sexual reproduction. In order to obtain a statistical significant sample size, 100

gametangia from each line were counted (using a stereoscope) and the number of sporophytes present

was determined. This number represents the percentage of fertilization events in that sample. A total of

8 counts were done per mutant line (Δdrm2#1 and Δdrm2#2) plus respective WT in the generation zero

(F0) and a total of 5 counts per mutant line plus the respective WT in the generation one (F1) and

generation two (F2). WT percentages below 40% were discarded (abnormal samples, Marcela

Coronado, personal communication) and replaced by new samples.

Genotyping of selection-surviving lines: Multiplex in-tissue PCR reactions were performed for

genotyping of the colonies obtained after selection of the protoplasts transformed according Cove (2005)

and Schaefer and Zrÿd, (2001) to obtain Δdnmt3b lines. In these reactions, tissue samples collected

into PCR reaction buffer were used as template DNA-containing samples. These multiplex reactions

used primers to check the presence/absence of the target gene, the resistance mark used to select the

mutant lines and the 5 and 3’ flanking regions of the target gene (used for homologous recombination).

Sporophyte collection, spore sterilization and germination: In order to obtain the F1 and F2 tissue

it was necessary to germinate the spores of the previous generation. For spore sterilization three mature

sporophytes were collected into a 1.5 mL eppendorf tube and transferred to a flow hood wherein, 1 mL

of 5 % bleach solution was added to the sporophytes and incubated at room temperature for 5 minutes.

The bleach solution was then removed from the tubes that were washed 3 to 4 times with 1 mL of sterile

water. Then 1 mL of sterile water was added to the sterile spore capsules. These were broken with a 1

mL pipette tip, releasing the spores into the water. The estimated concentration of spores was of 15

spores per microliter of water, and these were germinated in petri dishes with minimal media (KNOPS)

or stored at 4 ºC.

Colony growth assays: for the germination of the spores, 3 μL of sterilized spores-containing

water were added to a small water bottle containing 9 mL of sterile MiliQ water, mixed and then 3 mL

were distributed by each petri dish with minimal media. The plates with the spores were incubated at 25

ºC with 16 hours on light and 8 hours on dark daily cycles during 21 days. 21 days old colonies were

imaged using a Leica Stereoscope with a color camera. To germinate spores at different pH, bottles

with buffered water at pH values of 6.52, 6.9 and 7.51 contained 5 mM MOPS buffer while the bottles

with the water at pH 7.8, 8.28 and 8.93 were buffered with 5 mM of Tris-HCl.

For the colony area assay, sterilized spores were germinated every 2 weeks after being stored

on 4 ºC for 0 to 14 weeks. The growth of the colonies was followed by imaging of the colonies’

autofluorescence (Texas Red channel) using a Zeiss Stereo LUMAR stereoscope controlled with

MicroManager version 1.14 software, at different days of their growth: days 3, 5, 7, 10, 15 and finally

day 21 of growth when, and whenever possible, 25 colonies were picked for colony dry weight

assessment. Dry weight of the colonies was assessed by subtracting the paper weight from the final

microwave dried colony sample weight. Image analysis was performed using imageJ software.

Statistical analysis: Mann-Whitney t-tests and Kruskal-Wallis’ one-way analysis of variance

(ANOVA) followed by Dunn’s multiple comparison tests were performed in Prism5 (GraphPad) software.

Statistical significance of the differences was evaluated considering a 95 % confidence interval.

Antherozoid release and labeling: WT tissue was grown on jiffies for 3 weeks under long day

conditions, time after which we induced the sexual reproduction phase. 15 days later manual dissection

of antheridia (using a stereoscope) into 20 µL of sperm nutritive solution (supplemented with 10 µg/mL

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of fluoresceín diacetate (FDA) for labeling assays) was performed. The samples were then observed

under oil immersion with a 100x magnification, on a custom-built high-throughput setup, based on a

Nikon Eclipse TE2000-S equipped with a Hamamatsu Flash 2.8 sCMOS camera and controlled with the

MicroManager version 4.1.14 software. The labeled samples were observed under the same

microscope and with the same settings as the unlabeled samples (detailed before) but also in addition,

green fluorescent signal was observed on the green fluorescent protein (GFP) channel.

Flow cytometry and antherozoids cell sorting: mature antheridia samples were manually

dissected for 20 min into a 1.5 mL eppendorf tube with 100 µL of sperm nutritive solution. The sample

was filtered through a custom made column + filter assembly, wherein the filter used was a mesh filter

with either 10 µm or 28 µm of pore diameter, by centrifugation at 280 g for 1min. Next, another 100 µL

of sperm nutritive solution were added to the column and filter assembly and the centrifugation step was

repeated. This step was repeated again. These filtrations were designed to try to release the

antherozoids from the clusters and push them through the filter, in order to obtain them isolated in the

flow-through.

A portion of the flow-through sample was analyzed in a Modular Flow Cytometer (MoFlo) and the

cellular profile of the sample was determined (unstained sample). Then, 0.6 µL of FDA (2 mg/µL) was

added to the remaining sample and the flow cytometric analysis was repeated (stained sample). The

population of interest was identified by the absence of an autofluorescent (red) signal and the presence

of a FDA (green) signal. Part of this isolated population was sorted using the MoFlo and observed using

a Nikon Eclipse TE2000-S equipped with a Hamamatsu Flash 2.8 sCMOS camera and controlled with

the MicroManager version 4.1.14 software under oil immersion at 100x amplification, in order to confirm

the antherozoid presence in the analysed samples.

Results and Discussion

Differences in fertilization rate are only detected for Δdrm2#1 in the F0 generation

Due to the previous observation that the DRM2 gene is only significantly expressed in P. patens’

antherozoids, sexual reproduction could be affected due to inability of the antherozoids to fertilize the

egg cell or arrested zygote development. Therefore, the first step for the characterization of the Δdrm2

lines obtained previously (Δdrm2#1 and Δdrm2#2) was to assess their fertilization rates. This

assessment was performed both for the F0 generation - tissue that was transformed as well as for the

F1 and F2 generations - obtained by the germination of F0's and F1's spores respectively, for both mutant

lines as well as for the WT samples grown together with the mutant lines.

The scatter plots of the fertilization rates obtained for the each generation of the lines analysed in

this work (WT#1, WT#2, Δdrm2#1 and Δdrm2#2) as well as a summary of the t-tests performed are

shown on Figure 1. In the F0 generation (Figure 1 A), the average fertilization rate obtained for WT#1

line was of 63%, this value was 63% for WT#2, 47% for Δdrm2#1 and for Δdrm2#2 line was of 52%.

F1’s fertilization rates were 49%, 53%, 47% and 50% for WT#1, WT#2, Δdrm2#1 and Δdrm2#2,

respectively (Figure 1 B) and in the F2 generation average fertilization rates were 64% for WT#1, 61%

for WT#2, 63% for Δdrm2#1 and 57% for Δdrm2#2 (Figure 1 C). Results from the statistical analysis by

t-test revealed that the only statistically significant difference detected was between WT#1 and Δdrm2#1

lines in the F0 generation (p-value = 0.0006, Figure 1 A). The differences detected between Δdrm2#1

and Δdrm2#2 lines can be due to high standard deviations of both WT#2 and Δdrm2#2 lines, different

insertion’s copy and/or due to different ploidy levels (Schween et al., 2005).

No differences were detected in the F1 and F2 generations, meaning that, if any difference in the

fertilization between WT and DRM2 deletion rates exists, it’s only in the F0 generation, being restored

after the first fertilization event (Figure 1). A possible explanation for this is that the expression of

DNMT3b in the antherozoids may be sufficient to compensate for the deletion of DRM2.

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Figure 1: Fertilization rates from wild-type (WT) grown with line 1 (WT#1) and line 2 (WT#2) as well as

for DRM2 mutant lines (Δdrm2#1 and Δdrm2#2). A: Fertilization rates for generation zero (F0, n = 8); B: Fertilization rates of generation one (F1, n = 5); C: Fertilization rates of generation two (F2, n = 5). Round dots represent WT samples fertilization rate while triangles represent DRM2 mutant lines’ fertilization rates. Black horizontal bars represent the average of the lines’ fertilization rates and grey vertical lines represent samples standard error. Dashed horizontal lines represent the results of the Mann-Whitney’s t-tests performed. ns: non-significant differences; *** : p-value < 0.01, significant differences detected.

Colonies appearance shows phenotypic variations after cold storage of spores

In order to obtain and analyse the F1 generation of the WT and Δdrm2 lines, the germination of

F0 spores was required. After the sterilized spores were stored at 4 ºC during 14 weeks and allowed to

germinate, all 21 days-old WT colonies showed a more irregular shape with a few gametophores

developing (Figure 2 A) when compared to the colonies obtained from freshly sterilized spores (Figure

2 C), probably trying to expand and looking for better conditions to grow. Colonies of Δdrm2#2 line

showed a more regular round shape and were smaller than the WT ones (Figures 2 D). This effect

suggested some sort of problem in spore germination and/or colony growth after cold storage of spores,

with Δdrm2#1 spores being highly affected (maybe losing viability) and Δdrm2#2 spores more affected

than the WT’s, since these line’s spores didn’t germinate.

This led us to design a more exhaustive experiment where spores would be germinated

immediately after sterilization (freshly sterilized spores) and every 2 weeks after being stored at 4 ºC,

until 14 weeks of storage. In order to try to assess possible differences in the growth rate and final dry

weight of the colonies growth from spores of different lines and after different periods of cold storage,

total colony area was determined after 3, 5, 7, 10, 15 and 21 days of growth.

Unfortunately, from both the colonies’ area and their dry weight analyses, no biologically and

consistent relevant differences were detected between WT and Δdrm2 lines. Nevertheless, these

methods can now be used to study the protonema development in other lines.

Smaller colonies with a round regular shape were again observed during the detailed assay to

study colony growth performed, in samples from WT#1 after 6 weeks of cold storage of spores (3/10

plates) and in colonies of Δdrm2#1 line after 10 weeks of storage of spores at 4 ºC (2/4 plates). The

appearance of such colonies on both WT and Δdrm2 samples indicates that this is not due to any

particular effect of the DRM2 deletion. Due to the fact that the smaller and round colonies are always

detected on three plates and that one water bottle (with 9 mL of water) is used to spread the spores in

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3 plates, the idea that the altered shape of the colonies

could be due to differences in the water bottles arose.

All the bottles are always cleaned together and the

same treatment is applied to all. Therefore, we decided

to analyze the pH of the water in each bottle after

sterilization.

The pH of the water of 10 different bottles was

analyzed and it was found to vary between 6.9 and 8.9

(data not shown). The pH effects many cellular

processes, such as ionic exchanges, enzymatic

activities and compound solubility (Apinis, 1939), it

could have some sort of effect on the germination of

the spores and growth of the colonies. In order to test

this hypothesis, germination of spores with water at

different pH values was performed. It was possible to

obtain colonies in all samples meaning that spore were

able to germinate in all the different pH values tested.

This is in accordance with Apinis (1939), who had

already reported that spores from several bryophytes

could germinate in a wide range of pH values. No significant differences in colonies’ shape and aspect

were detected, indicating that water’s pH may not be the only reason influencing the phenotype of the

colonies. Similar phenotypes were already reported for mutants of genes related with auxin signaling

(AFB and IAA) (Prigge et al., 2010); Dof1 transcription factor mutants (Sugiyama et al., 2012);

RSL1/RSL2 transcription factor double (Jang and Dolan, 2011) and PIPK1 and PIPK1 PIPK2 K.O. lines

(Saavedra et al., 2011). So far, no connection between the variation in colony growth detected in this

work and prolonged cold storage of similar phenotypes reported could be established.

Δdnmt3b knockout lines were not obtained

To explore the hypothesis of DNMT3b compensating the DRM2 deletion in the antherozoids, PEG

mediated protoplast transformation of the WT line with pSP3b plasmid was performed, with the purpose

of obtaining Δdnmt3b lines. Two rounds of selection followed the transformation and only 10 colonies

were obtained after selection. In-tissue multiplex (Figure 3) were used to genotype the selection

surviving colonies, using a WT tissue sample as a positive control for the WT genotype.

Reactions C used primers specific for DNMT3b gene and primers annealing in the mCherry gene

present in the plasmid used in the transformation. The expected sizes for the amplified fragments in the

reactions C were of 2137 nt, if the DNMT3b gene is present on the sample used as template DNA (WT

samples) and 1292 nt in the cases where the DNMT3b gene was efficiently replaced by the mCherry

coding sequence (Δdnmt3b lines). Reactions D included primers specific for DNMT3b gene as well as

a primer annealing on the genomic DNA region near the HR sites, on both WT and mutant samples and

another specific for the hygromycin B resistance gene, only present if the homologous recombination

was successful. Reactions D should result in the amplification of a DNMT3b gene fragment with 883 nt,

in the lines where the gene was not replaced (WT genotype) and a fragment with 1289 nt on Δdnmt3b

lines.

As observable from Figure 3, from both C and D reactions, the only fragments obtained had

similar sizes to the ones obtained from the WT tissue sample (positive control, Figure 3 reactions C WT

and D WT samples). The band detected in samples from reactions C has around 2100 nt (Figure 3,

Figure 2: Colonies after 21 days of growth.A: F0’s wild-type colonies obtained from freshly sterilized spores. B: colonies of Δdrm2#2 F0 from freshly sterilized spores. C: F0’s wild-type colonies obtained from spores stored at 4 ºC for 14 weeks. D: colonies of Δdrm2#2 F0 from spores stored at 4 ºC for 14 weeks. Scale bars = 1 mm.

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reactions C #1-10), while from reactions D the bands detected have between 800-900 nt (Figure 3,

reactions D #1-10). This indicates that all selection-surviving colonies regenerated from the protoplasts

subjected to transformation represent WT genotype colonies and that no Δdnmt3b were lines generated.

Figure 3: 1 % agarose gel loaded with the in-tissue multiplex PCRs C and D, used for genotyping the

selection-surviving colonies from the transformation of Physcomitrella patens protoplasts with the pSP3b plasmid. 1kb ladder (NEB) was used to estimate the amplified fragments size. The size of the ladder’s bands are presented on the left part of the image. Wells numbered #1 to #10 represent the selection-surviving colonies number and the WT samples represents the reactions using wild-type tissue as template DNA (positive control).

Antherozoids lack autofluorescence and can be labeled with FDA

Figure 4: FDA labelled antherozoid. Mature antheridia were manually dissected into sperm nutritive solution

supplemented with fluorescein diacetate (FDA) and the antherozoids were released. Microscopic observation was achieved at 100x magnification under oil immersion. A: widefield image (15 ms of exposure time); B: green fluorescent signal detected (100 ms exposure time); C: red fluorescent signal – autofluorescence, detected in the red mCherry fluorescent protein channel (250 ms exposure time); D: merged image of the observed antherozoid in widefield, green and red channels. Scale bars = 10 μm.

With the goal of developing a more time-efficient method to collect P. patens antherozoids,

namely by the use of FACS, we first had to label these cells. The fluorescent labelling of the antherozoids

was achieved by adding FDA to the sperm-nutritive solution in which the mature antheridia were

dissected and the antherozoids released. Afterwards, the samples were observed under oil immersion

at 100x magnification in order to detect if release of the antherozoids clusters had occurred and if they

were labelled. In Figure 4 A, a labelled isolated antherozoid in bright field is shown, green fluorescence

(Figure 4 B) present in both the antherozoid body and its flagella, as well as the absence of

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autofluorescence, analysed by red fluorescence using the red channel (Figure 4 C). The merged picture

of all three channels can be seen in Figure 4 D.

FACS sorting of antherozoids.

After successful labelling of intact antherozoids with FDA, the next step in the development of a

time-efficient protocol to collect the antherozoids was to test if sorting of these cells was possible by an

automated process, such as FACS.

In order to allow the analysis of samples by flow cytometry, the particles in the sample could not

be bigger than 50 µm in diameter. Therefore, mature antheridia and antherozoids clusters had to be

filtered out from the sample before FACS. 10 µm mesh and 28 µm mesh filters were tested, and isolated

antherozoids were observed in the flow-through solutions obtained after the samples’ filtering with either

type of mesh. When the sperm-nutritive solution was supplemented with FDA, the isolated antherozoids

showed a bright green and no significant red fluorescent signals.

Next, new samples were prepared into sperm-nutritive solution and analyzed in the MoFlo cell

sorter. After filtration, a portion of flow through solution was examined - unstained sample. Then, FDA

was added to the remaining sample - FDA stained sample. The same analysis performed on part of the

unstained sample was repeated for the stained sample. Our flow cytometry results allowed the

identification of a population with the characteristics of interest (green positive and red negative signals)

in the stained samples, part of this population was sorted and microscopic confirmation of the presence

of antherozoids in the sorted sample was successful (Figure 5).

This novel method to collect isolated and viable antherozoids of P. patens will still require further

optimization, so that a high number of isolated antherozoids can be obtained in the shortest time

possible.

Figure 5: Antherozoids sorted by FACS. Images obtained by microscopic observation of the sorted part of

the population of interest, isolated by FACS of the sample examined in Figure 35. A total of 289 events were sorted. In each image isolated antherozoids are shown, and on the right image some debris can also be observed (red label). Scale bars = 5 µm.

Concluding Remarks

In conclusion, we were unable to detect any consistent defect in the Δdrm2 lines analysed,

possibly due to dnmt3b compensation, whose deletion lines could not be obtained in the course of this

work. A link between the different aspect of the colonies described in this work, and the similar reported

phenotypes could not be established. However, methods to analyse colony growth by colonies area and

dry weight, as well as genotyping by in tissue PCR were successfully implemented during the

progression of this work.

Moreover, this study describes a novel automated method to efficiently sort antherozoids of P.

patens by FACS.

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