# Transcriptomics of manually isolated Amborella trichopoda egg apparatus cells

María Flores-Tornero, Sebastian Proost, Marek Mutwil, C.P. Scutt, Thomas

Dresselhaus, Stefanie Sprunck

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► To cite this version:

María Flores-Tornero, Sebastian Proost, Marek Mutwil, C.P. Scutt, Thomas Dresselhaus, et al. *Transcriptomics of manually isolated Amborella trichopoda egg apparatus cells*. Sexual Plant Reproduction, 2019, 32 (1), pp.15-27. 10.1007/s00497-019-00361-0. hal-02348736

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# Transcriptomics of manually isolated *Amborella trichopoda* egg apparatus cells

María Flores-Tornero<fnref n="1" /> · Sebastian Proost<fnref n="2" />,<fnref n="5" /> · Marek Mutwil<fnref n="2" />,<fnref n="3" /> · Charles P. Scutt<fnref n="4" /> · Thomas Dresselhaus<fnref n="1" /> ·
Stefanie Sprunck<fnref n="1" />

Received: 7 November 2018 / Accepted: 9 January 2019 / Published online: 1 February 2019
© The Author(s) 2019, corrected publication 2019

**Key message** A protocol for the isolation of egg apparatus cells from the basal angiosperm *Amborella trichopoda* to generate RNA-seq data for evolutionary studies of fertilization-associated genes.

**Abstract** Sexual reproduction is particularly complex in flowering plants (angiosperms). Studies in eudicot and monocot model species have significantly contributed to our knowledge on cell fate specification of gametophytic cells and on the numerous cellular communication events necessary to deliver the two sperm cells into the embryo sac and to accomplish double fertilization. However, for a deeper understanding of the evolution of these processes, morphological, genomic and gene expression studies in extant basal angiosperms are inevitable. The basal angiosperm *Amborella trichopoda* is of special importance for evolutionary studies, as it is likely sister to all other living angiosperms. Here, we report about a method to isolate *Amborella* egg apparatus cells and on genome-wide gene expression profiles in these cells. Our transcriptomics data revealed *Amborella*-specific genes and genes conserved in eudicots and monocots. Gene products include secreted proteins, such as small cysteine-rich proteins previously reported to act as extracellular signaling molecules with important roles during double fertilization. The detection of transcripts encoding EGG CELL 1 (EC1) and related prolamin-like family proteins in *Amborella* egg cells demonstrates the potential of the generated data set to study conserved molecular mechanisms and the evolution of fertilization-related genes and their encoded proteins.

**Keywords** Egg cell · Synergid cell · Microdissection · EC1 · RALF · RNA-seq · *Amborella*

## Introduction

Molecular and fossil evidence indicates that flowering plants (angiosperms) arose abruptly in the late Jurassic/early Creta- ceous from a not yet identified ancestral lineage. Since then, angiosperms rapidly diversified to form over 350,000 species

alive today (Bell et al. 2005; Doyle 2012; Willis and McEl- wain 2013; Scutt 2018) and represent the most abundant and ecologically successful group of plants on earth. *Amborella trichopoda*, a woody shrub endemic to New Caledonia, holds a key position in the angiosperm evolutionary tree as the most basal angiosperm. It is the sole living species of the Amborellaceae family which is strongly supported as the sister lineage of all other extant flowering plants (Amborella Genome Project 2013). For this reason, *Amborella* has been the focus of many phylogenetic, genomic and reproductive biology studies, providing a deeper understanding regarding the evolution of flowering plants (for review, see Scutt 2018).

A contribution to the special issue ‘Cellular Omics Methods in Plant Reproduction Research’.

Communicated by Dolf Weijers.

**Electronic supplementary material** The online version of this article (https://doi.org/10.1007/s00497-019-00361-0) contains supplementary material, which is available to authorized users.

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Stefanie Sprunck
stefanie.sprunck@ur.de

<footnote n="1">¹ Cell Biology and Plant Biochemistry, Biochemie-Zentrum Regensburg, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany</footnote>

<footnote n="2">² Max-Planck Institute for Molecular Plant Physiology, Am Muehlenberg 1, 14476 Potsdam, Germany</footnote>

<footnote n="3">³ School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore</footnote>

<footnote n="4">⁴ Laboratoire Reproduction et Développement des Plantes, École Normale Supérieure de Lyon, Université Claude Bernard Lyon 1, CNRS, INRA, Université de Lyon, Lyon, France</footnote>

<footnote n="5">⁵ Present Address: Laboratory of Molecular Bacteriology (Rega Institute), KU Leuven, Louvain, Belgium</footnote>
Among the defining features of angiosperms are carpels, which enclose and protect the ovules, and the process of double fertilization involving two pairs of male and female gametes, respectively (Dresselhaus et al. 2016; Scutt 2018). The highly reduced female gametophyte (embryo sac) develops within the ovule and contains the egg cell and central cell. Both cells become fertilized and give rise to the embryo and the embryo-nourishing endosperm of the seed, respectively. A large majority of extant angiosperms produce the monosporic *Polygonum*-type embryo sac, which was first described in *Polygonum divaricatum* (Strasburger 1879). This originates from a single haploid spore (the functional megaspore), which undergoes three incomplete mitotic division cycles to develop into an eight-nucleate syncytium. After cellularization, the *Polygonum*-type embryo sac is seven celled but eight nucleate, with three cells at each pole and a large binucleate central cell. The egg apparatus is located close to the micropylar entrance point of the pollen tube and comprises one egg cell and two accessory synergid cells, which are required for pollen tube attraction and reception (for review, see Higashiyama and Yang 2017). Three antipodal cells form at the opposite chalazal end of the embryo sac, but little is known about their function (Sprunck and Groß-Hardt 2011).

Although the *Polygonum*-type embryo sac is most prevalent in flowering plants, embryo sac formation is highly diverse across different plant taxa, suggesting an extensive degree of developmental experimentation during angiosperm evolution (Friedman 2006). Embryo sacs can also develop from two or four functional megaspores (bisporic and tetrasporic development, respectively), and/or from a varying number of mitotic divisions following meiosis. Within the embryo sac, the cells can furthermore occupy different positions (Maheshwari 1950; Huang and Russell 1992).

Notably, none of the members of the earliest extant angiosperm clades produces a *Polygonum*-type embryo sac (Friedman 2006). The four-celled *Nuphar/Schisandra*-type embryo sac comprising one egg cell, two synergid cells and a uninucleate central cell appears to be universal among extant members of the Nymphaeales and occurs also in Austrobaileyales (e.g., Williams and Friedman 2004; Tobe et al. 2007; Friedman 2008; Rudall et al. 2008). According to the present knowledge, the nine-nucleate, eight-celled *Amborella*-type female gametophyte is restricted to *Amborella* (Friedman and Ryerson 2009) and might represent a critical link between angiosperms and gymnosperms (Friedman 2006). Detailed structural analyses revealed that *Amborella* embryo sac development initially parallels that of the *Polygonum*-type, since immature embryo sacs are eight nucleate and seven celled, with each three cells at the chalazal and the micropylar pole. However, prior to embryo sac maturation, one of the three cells at the micropylar pole undergoes a final mitotic and cytokinetic division to produce two daughter

cells: a third synergid cell and the egg cell. At maturity, the *Amborella* embryo sac is therefore eight celled, with three antipodal cells, a large binucleate central cell and a unique four-celled egg apparatus comprising three synergid cells and the egg cell which is the lineal sister cell of a synergid cell (Friedman 2006).

From studies in model plants such as *Arabidopsis* and maize, we know that cell fate specification processes in the developing embryo sac are tightly controlled and a few of the genes involved in these have been identified (for review, see Tekleyohans et al. 2017; Zhou et al. 2017). Previous studies furthermore showed that synergid cells, egg cells and central cells fulfill unique functions during flowering plant reproduction, which are reflected by distinct gene expression profiles (Márton et al. 2005; Okuda et al. 2009; Wuest et al. 2010; Anderson et al. 2013; Chen et al. 2017). These include molecules mediating pollen tube guidance to the ovule, induction of pollen tube burst, gamete interaction, activation and fusion (Okuda et al. 2009; Amien et al. 2010; Márton et al. 2012; Sprunck et al. 2012; Takeuchi and Higashiyama 2012; Ge et al. 2017). Nevertheless, a deeper molecular understanding of the evolution of flowering plant embryo sac formation is missing, and so far, the transcriptomes of individual embryo sac cells from basal angiosperms remained unexplored.

Here, we describe a method to manually isolate viable *Amborella* egg apparatus cells. Their morphology was investigated, and they were subsequently used to generate genome-wide gene expression profiles from a low number of cells for bioinformatics analysis. In this pioneer study, we identified *Amborella*-specific genes preferentially expressed in egg apparatus cells and first candidate genes for comparative evolutionary and functional studies.

# Materials and methods

## Plant material and growth conditions

Flowers were harvested from a female *Amborella* plant (one flowering period per year), grown in the greenhouse under controlled conditions of 12 h of light, 16.2 °C and constant humidity of 66%. Control tissues of tepals, leaves and roots were harvested and immediately frozen in liquid nitrogen.

## Isolation of egg apparatus cells

The isolation procedure was performed only on the most recently fully opened flowers with visible wet stigmas, harvested in the morning (8:00 am). Flowers were harvested in a glass petri dish on wet filter paper, to keep high humidity until dissection. All following steps were performed at room temperature, without any breaks and in an expeditious manner (approximately 1.5 h for processing ten carpels). Under

<figure><img src="image_4.png" /></figure>
a Nikon ZM645 stereo microscope, individual carpels were gently removed from the inflorescence using disposable hypodermic needles (0.4 mm diameter, 13 mm length) and placed in a new glass petri dish. Subsequently, carpels were cross-sectioned using a scalpel (No. 11 sterile carbon steel surgical blade). Cutting angle and position of cross section is indicated in Fig. 1b. The lower portion of dissected carpels is immediately transferred to a microscopy Imaging Dish GC 1.5 (130-098-284, Miltenyi Biotec GmbH), filled with 0.3 M filter-sterilized mannitol solution. Subsequently, hypodermic needles (0.4 mm diameter, 13 mm length) were used to further dissect the lower portion of the carpel in mannitol solution. The inner integument was separated from the outer integument, and the inner part of the ovule was gently lifted out of the carpel. The remains of the outer ovary sections were removed. Subsequently, the release of egg apparatus cells from the ovule tips was performed under a Nikon Eclipse TE2000-S inverted microscope equipped with a 20× objective (Plan Fluor ELWD 20×/0.45, DIC L/N1). For the isolation procedure, fine-tipped glass needles were prepared from borosilicate glass rods (4 mm diameter, 10 cm length, Hilgenberg GmbH) by using

a heating device (Bunsen burner). To release egg apparatus cells from the ovule tip, one glass needle was used to hold the inner integument in place. The cells were then released by gently tapping on the outer side of the ovule with the second glass needle. Once egg apparatus cells were released, and their diameter was recorded by using a 40× objective (Plan Fluor ELWD 40×/0.60, DIC L/N1, Nikon). Cells were immediately collected using a CellTram® Oil (Eppendorf) equipped with a Ringcaps® glass 50-micron capillary (Hirschmann Instruments™ 9,600,150). The fine tip of the glass capillary was prepared using a micropipette puller as described elsewhere (Englhart et al. 2017). Individual cells were transferred to 0.5 ml LoBind reaction tubes (Sigma Aldrich) in a minimal amount of mannitol solution (max. 3–5 µl) and immediately frozen in liquid nitrogen for further analyses. Frozen cells were stored at –80 °C.

RNA isolation, library preparation and sequencing

For control tissues, total RNA was isolated from three biological replicates of tepals, leaves and roots with a

<figure><img src="image_5.png" /><figcaption>**Fig. 1** Female flower of *A. trichopoda* and isolation procedure of egg apparatus cells. **a** Female flower at anthesis. **b** Longitudinal cut of a mature carpel (side view). Note that the abaxial side of the carpel is more curved, while the adaxial side is flat. The red dashed line shows the angle of the cross section through the carpel. The position of the egg apparatus cells (asterisk) within the embryo sac (dotted line) is indicated. **c** Scheme of the Amborella embryo sac. Note that the cross section (red dotted line) traverses the lumen of the central cell. **d** Lower portion of the dissected carpel in mannitol solution. The dashed lines border the ovules outer integument. The arrowhead marks the position where the inner integument is manually separated from the outer integument and lifted out of the carpel. **e** Character-</figcaption></figure>

istic appearance of manually dissected ovule tip with the lower portion of the embryo sac (dotted line), surrounded by nucellus and inner integument. **f** Dissected ovule tip releasing an egg apparatus cell. **g** Morphology of released egg apparatus cell. The fluorescent dyes SYBR Green I and FM 4–64 were applied to stain the nucleus (green) and the plasma membrane (red), respectively. AC, antipodal cells; ad, adaxial side of carpel; CC, central cell; Cp, carpel; EAC, egg apparatus cell; EC, egg cell; ES, embryo sac; M, micropyle; N, nucleus; Ov, ovule; PM, plasma membrane; PN, polar nuclei; S, stigma; St, staminoide; Sy, synergids; T, tepal; V, vacuole. Scale bars in **a**, **b**: 1 mm; in **d**, **e**: 100 µm; in **f**: 25 µm; in **g**: 10 µm
Spectrum™ Plant Total RNA Kit (Sigma Aldrich) according to manufacturer's instructions. RNA quality was assessed on the Agilent 2100 Bioanalyzer with the RNA 6000 Pico LabChip reagent set (Agilent) (Supplementary Figure 1). RNA integrity (RIN) values ranged from 6.7 to 9.4. Using 250 ng of total RNA, library preparation and RNA-seq were carried out as described in the Illumina TruSeq Stranded mRNA Sample Preparation Guide, the Illumina HiSeq 1000 System User Guide (Illumina, Inc.) and the KAPA Library Quantification Kit—Illumina/ABI Prism User Guide (Kapa Biosystems, Inc.).

Total RNA from egg apparatus cells was extracted according to the “Purification of total RNA from animal and human cells” protocol of the RNeasy Plus Micro Kit (Qiagen). Twenty-eight cells of L-(diameter from 25.85 to 17.96 µm) and M-(diameter from 17.65 to 16.35 µm) size categories and 26 cells of the S-size category (diameter from 16.17 to 13.13 µm) were used for RNA extraction. Finally, total RNA was eluted in 12 µl of nuclease-free water. RNA quality control measurements on the Bioanalyzer were unsuitable, since the RNA quantities obtained from the low number of egg apparatus cells were below the assay sensitivity. The SMARTer Ultra Low Input RNA Kit for Sequencing v4 (Clontech Laboratories, Inc.) was used to generate first-strand cDNA from 500 pg total RNA. Double-stranded cDNA was amplified by LD PCR (14 cycles) and purified via magnetic bead cleanup. Library preparation was carried out as described in the Illumina Nextera XT Sample Preparation Guide (Illumina, Inc.). 150 pg of input cDNA was tagmented (tagged and fragmented) by the Nextera XT transposome. Products were purified and amplified via a limited-cycle PCR program to generate multiplexed sequencing libraries. For the PCR step, 1:5 dilutions of index 1 (i7) and index 2 (i5) primers were used.

Libraries from egg apparatus cells and control tissues were quantified using the KAPA SYBR FAST ABI Prism Library Quantification Kit. Equimolar amounts of each library were used for cluster generation on the cBot (TruSeq SR Cluster Kit v3). Sequencing runs were performed on a HiSeq 1000 instrument using the indexed, 2×100 cycles paired end (PE) protocol and the TruSeq SBS v3 Kit. Image analysis and base calling resulted in “.bcl” files, which were converted into “.fastq” files using the CASAVA1.8.2 software. RNA extraction of egg apparatus cells and Illumina deep sequencing were carried out at the genomics core facility of the University of Regensburg (Center for Fluorescent Bioanalytics KFB; www.kfb-regensburg.de).

## RNA-seq data analysis

RNA-seq data were processed using Kallisto (Bray et al. 2016). Reads were mapped to the *Amborella* genome v1.0 (Amborella Genome Project 2013), obtained from Ensembl Plants (Kersey et al. 2016). Gene expression values were

determined using Transcripts Per Million (TPM) (Conesa et al. 2016) and clustered using Python Seaborn software package with “Euclidean” distance and “complete” method, which was also used to generate the heat map shown in Fig. 3c. A comprehensive overview on normalized expression data in *Amborella* egg apparatus cells, leaves, roots and tepals is shown in Supplementary Table 1. RNA-seq data will be publicly available in the open source web server CoNekT (Proost and Mutwil 2018).

## Results and discussion

### Isolation of *Amborella* egg apparatus cells

We report here an isolation procedure for *Amborella* egg apparatus cells by manually dissecting carpels of female flowers, without treating ovules with cell wall-degrading enzymes. Enzyme-free methods have been successfully established mainly for the isolation of egg cells from angiosperm species such as *Brassica napus*, *Hordeum vulgare*, *Oryza sativa*, *Plumbago zeylanica* and *Triticum aestivum* (Table 1). After gently removing carpels from female flowers of *Amborella* (Fig. 1a), their characteristic shape with a flat adaxial and round abaxial side became apparent. Carpels were placed laterally to perform a cross section through ovaries as indicated in Fig. 1b. It was very clear in our studies that position and angle of cross sections determined later success of releasing egg apparatus cells. Cross sections traverse the lumen of central cells (Fig. 1c), which will be destroyed and, thus, cannot be isolated by the described protocol. Immediate transfer of the lower part of carpels into 0.3 M mannitol solution is essential to prevent osmotic stress and to avoid the irreversible collapse of egg apparatus cells by drying out (Fig. 1d). In pilot experiments, we tested different mannitol molarities and determined 0.3 M mannitol to be optimal to maintain the shape and size of released egg apparatus cells. Similar mannitol concentrations have been reported for the isolation of egg apparatus cells from rice and spider lily (Uchiumi et al. 2006; Ohshika and Ikeda 1994). By contrast, egg apparatus cells from other flowering plant species require higher osmolaries, such as 0.55 M mannitol for wheat (Kovács et al. 1994), or even 0.7 M mannitol for *Plumbago zeylanica* and rapeseed (Cao and Russell 1997, Katoh et al. 1997). We used one hypodermic needle to hold the outer part of the carpel in place. With a second hypodermic needle, we gently separated the inner integument from the outer integument and lifted ovule tips out of carpel sections (Fig. 1e). After removing remains of carpels from the mannitol solution, release of egg apparatus cells was performed and observed under an inverted microscope. Two fine-tipped glass needles were used to hold one ovule tip and to gently push the egg apparatus cells out of the tip, respectively. One egg apparatus cell, just being released

<figure><img src="image_6.png" /></figure>
<table><caption>**Table 1** Angiosperm species whose female gametophytic cells have been successfully isolated from living material</caption><thead><tr><th>Organism</th><th>EC</th><th>SY</th><th>CC</th><th>Method</th><th>References</th></tr></thead><tbody><tr><td>Alstroemeria aurea</td><td>50.4</td><td>45.2</td><td>—</td><td>E/M</td><td>Hoshino et al. (2006)</td></tr><tr><td>Amborella trichopoda</td><td>18–25</td><td>13–15</td><td>—</td><td>M</td><td>This study</td></tr><tr><td>Arabidopsis thaliana</td><td>13–15</td><td>16–17</td><td>24–26</td><td>E</td><td>Englhart et al. (2017)</td></tr><tr><td>Brassica napus</td><td>10–12</td><td>—</td><td>18–25</td><td>M</td><td>Katoh et al. (1997)</td></tr><tr><td>Ceiba speciosa</td><td>13–15*</td><td>10–13*</td><td>—</td><td>E/M</td><td>Lin et al. (2012)</td></tr><tr><td>Crinum asiaticum</td><td>75–80*</td><td>80–100*</td><td>—</td><td>E/M</td><td>Ohshika and Ikeda (1994)</td></tr><tr><td>Datura stramonium</td><td>18–20*</td><td>14–16*</td><td>—</td><td>E/M</td><td>He et al. (2012)</td></tr><tr><td>Hordeum vulgare</td><td>30–33*</td><td>—</td><td>—</td><td>M</td><td>Holm et al. (1994)</td></tr><tr><td>Lolium perenne</td><td>50–60</td><td>—</td><td>—</td><td>E/M</td><td>van der Maas et al. (1993)</td></tr><tr><td>Nicotiana alata</td><td>22–24*</td><td>18–20*</td><td>70–73*</td><td>E/M</td><td>Huang et al. (1992)</td></tr><tr><td>Nicotiana tabacum</td><td>30–34*</td><td>40–43*</td><td>120–125*</td><td>E/M/S</td><td>Hu et al. (1985)</td></tr><tr><td></td><td>30*</td><td>40*</td><td>—</td><td>E/M</td><td>Tian and Russell (1997)</td></tr><tr><td>Oryza sativa</td><td>40–50</td><td>40–50*</td><td>100–120</td><td>M</td><td>Uchiumi et al. (2006)</td></tr><tr><td>Petunia hybrida</td><td>10–12*</td><td>10–12*</td><td>25–30*</td><td>E</td><td>Van Went and Kwee (1990)</td></tr><tr><td>Plumbago zeylanica</td><td>60–90</td><td>42–45<sup>1,*</sup></td><td>—</td><td>M</td><td>Cao and Russell (1997)</td></tr><tr><td>Solanum verbascifolium</td><td>30–32*</td><td>25–28*</td><td>90–92*</td><td>E/M</td><td>Yang et al. (2015)</td></tr><tr><td>Torenia fournieri</td><td>25*</td><td>18.7*</td><td>64.7*</td><td>E/M</td><td>Mól (1986)</td></tr><tr><td>Triticum aestivum</td><td>23–25*</td><td>18–19*</td><td>64–65*</td><td>E/M</td><td>Chen et al. (2008)</td></tr><tr><td></td><td>50–70</td><td>28–30*</td><td>—</td><td>M</td><td>Kovacs et al. (1994)</td></tr><tr><td>Zea mays</td><td>65–70</td><td>40–45</td><td>140–145</td><td>E/M</td><td>Kranz et al. (1991)</td></tr></tbody></table>

Average cell diameters of egg cells (EC), synergid cells (SY) and central cells (CC) in the respective cell isolation medium are indicated. References either report for the first time about the isolation of female gametophytic cells from a given species or provide a significantly improved method. E, enzymatic digestion; M, manual microdissection; S, gentle squashing. Average cell diameter (µm)

*Size inferred from images provided in the publication that is listed as a reference. ¹ Lateral cells, not synergid cells

from an ovule tip, is shown in Fig. 1f. Subsequently, egg apparatus cells were imaged, transferred into a 0.5 ml reaction tube by micropipette aspiration and immediately frozen in liquid nitrogen. Approximately 5–10 apparently intact egg apparatus cells could be isolated from 50 carpels.

## Morphology and identity of isolated egg apparatus cells

Depending on the plant species, either egg or synergid cells were reported to be slightly larger in diameter (Table 1). Only rice and petunia egg apparatus cells were reported to be rather similar in size (Van Went and Kwee 1990; Uchiumi et al. 2006). Furthermore, egg cells and synergid cells isolated from Arabidopsis, or rice, showed distinct cellular morphologies (Englhart et al. 2017; Ohnishi et al. 2011). To be able to distinguish *Amborella* egg cells and synergid cells without the help of molecular markers, we studied the morphology of isolated egg apparatus cells in more detail. Typically, intact cells exhibited a plasma membrane, a vacuole of variable size and a nucleus, which could be stained by SYBR Green I (Fig. 1g). Comparable to previous reports on *Plumbago*, wheat, barley and rice egg cells that were isolated by non-enzymatic cell isolation techniques (e.g.,

Holm et al. 1994; Cao and Russell 1997; Sprunck et al. 2005; Leljak-Levanic et al. 2013; Ohnishi et al. 2011), egg apparatus cells from *Amborella* became immediately round during the isolation process, suggesting that either a very thin primary cell wall or no cell wall was present.

Eventually, a group of spherical cells was simultaneously released from one ovary tip (Fig. 2). These cells exhibited slightly different morphologies in terms of cell size and granular, contrast-rich structures in their cytoplasm (Fig. 2a). The diameter of “large” cells ranged from 18 to 25 µm (±2.23), and they were frequently attached to either one, two or three smaller cells (Fig. 2a, b, d). The cytoplasm of these “large” cells, which we considered as egg cells (see below), contained relatively few granular particles and a large vacuole that pushed the nucleus (with its clearly recognizable nucleolus) to the cell periphery (Fig. 2d). Diameters of “small” egg apparatus cells ranged from 13 to 15 µm (±1.28). Furthermore, these cells featured less vacuoles, more granular particles in the cytoplasm and were sometimes released in a group of three (Fig. 2c). Based on their similar morphology and the fact that the female gametophyte of *A. trichopoda* contains three synergid cells (Friedman and Ryerson 2009), we determined the cells with diameters ranging from 13 to 15 µm as synergid cells.
<figure><img src="image_7.png" /><figcaption>**Fig. 2** Morphology of isolated egg apparatus cells from *A. trichopoda*. **a** Four spherical cells are released from the dissected embryo sac. Three cells appear slightly darker and smaller (arrowheads). **b** Group of one larger and two smaller cells that remain together after isolation. Two cells exhibit a clearly visible vacuole (V). **c** Group of</figcaption></figure>

Eventually, we observed clusters of egg apparatus cells where one of the three synergid cells was larger than the other two (Fig. 2a). Furthermore, a substantial number of isolated egg apparatus cells did not exhibit unambiguous morphological features and cell diameters, which prevented us from classifying them either as egg cells, or as synergid cells. However, the *Amborella* egg cell is unique in that it differentiates very late, only after a terminal cell division of one of the three micropylar cells of the seven-celled, eight-nucleate immature embryo sac present in pre-anthesis floral buds (Friedman 2006). The *Amborella* egg cell is therefore a lineal sister of the third synergid cell, while in all other angiosperm embryo sacs, the egg cell is the mitotic sister of a polar nucleus of the central cell (Huang and Russell 1992; Friedman 2006). Considering this uniqueness, the close relation between the egg cell and the third synergid cell of *Amborella* might be reflected by a more similar morphology, especially when the two daughter cells are isolated rather short after the final cell division. For these reasons, we assumed that the “intermediate-sized” and less vacuolated group of egg apparatus cells include young egg cells and their mitotic sister synergid cells, which cannot be unequivocally distinguished by their size and/or morphology. To obtain high-quality RNA-seq data, we had to pool at least 25 cells per replicate. We therefore assigned three egg apparatus cell size categories, based on the cell diameter (Fig. 3a). We pooled 28 cells with a diameter ranging from 17.96 to 25.85 µm as “Large” (L) group predominantly comprising egg cells. A group of 28 cells with diameters of 16.35 to 17.65 µm was classified as “Medium” (M) group enriched in young egg cells and third synergid cells. 26 cells with a diameter ranging from 13.13 to 16.17 µm were assigned as “Small” (S) group predominantly consisting of synergid cells.

three cells with similar size and morphology. **d** Two released cells showing different features in terms of size and cell morphology. The larger cell exhibits a clearly visible nucleolus (N) and a vacuole (V), while the smaller cell lacks a large vacuole and contains dark granules in the cytoplasm. Scale bars: 10 µm

RNA-seq analysis of isolated embryo sac cells

After RNA extraction, library preparation and Illumina sequencing, we obtained a total of 57.58 million (M) reads for the three groups of egg apparatus cells that mapped to Amborella genome v1 (Amborella Genome 2013). We also generated RNA-seq data from sporophytic control tissues (three biological replicates of leaves, roots and tepals, respectively) and obtained a total number of 22.62 M mapped reads for leaves, 19.07 M for roots and 50.38 M for tepals. Supplementary Table 1 provides *Amborella* gene symbols and normalized transcripts per million (TPM) values obtained for the three egg apparatus cell samples and the sporophytic control tissues.

A principal component analysis (PCA) performed on the expression data revealed that the three egg apparatus samples group closely together and are clearly separated from the clusters formed by the biological replicates of the three control tissues (Supplementary Figure 2). Among the egg apparatus cell size categories, L- and M-size cell types grouped together more closely.

We employed a threshold level of ≥ 1 TPM to estimate the number of expressed genes in the three groups of egg apparatus cells. Thereby, we detected 11,841 expressed genes in the category of large (L) cells, 11,808 expressed genes in the intermediate-sized cell category (M) and 12,501 expressed genes in the group of small (S) egg apparatus cells (Fig. 3b). Overall, 15,409 genes were expressed in the *Amborella* egg apparatus cells. Considering a total number of 27,313 coding genes in the genome of *A. trichopoda* (Ensembl Plants database release 41; September 2018), the number of egg apparatus-expressed genes comprises 56.42% of all *Amborella* genes.

When comparing gene expression patterns in the three cell size categories, we detected 8737 genes to be conjointly expressed in large, medium and small egg apparatus cells

<figure><img src="image_8.png" /></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 3** Size categories of *A. trichopoda* egg apparatus cells and overview of RNA-seq data generated. **a** According to their diameter, 82 isolated egg apparatus cells were pooled into groups of large (L), medium (M) and small (S) cells. **b** Number of expressed genes (TPM≥1) in each cell each size category and overlap (%) of the total number of expressed genes (15,409) among the three categories. **c**</figcaption></figure>

Heat map comparing the top 6000 genes expressed in each cell size
category. Genes are present in rows, while samples are present in col-
umns. Z-score normalized values are shown, ranging from 0 (mean
expression, in white) to 1 (maximum standard deviation away from
the mean of expression, in red)

(Fig. 3b and Supplementary Table 2). Despite this rather
large overlap (56.7% of 15,409 egg apparatus-expressed
genes), a comparison of the top 6000 genes expressed in
each cell size category identified prominent gene clusters
with differential expression in either the L-, M- or S-group
of egg apparatus cells (Fig. 3c). These distinct transcriptome
signatures could reflect a cell type-dependent accumulation
of transcripts necessary for the egg cells and synergid cells
to fulfill their specialized roles during double fertilization.
However, more biological replicates are needed to analyze
the differential gene expression in *Amborella* egg apparatus
cell types in detail, as only single replicates of each cell size
category have been generated during this pioneer study.

Egg apparatus-enriched genes encode CRPs, novel
proteins and proteins with homologs in eudicots
and/or monocots

We next analyzed the strongest expressed genes that were
specifically enriched in the respective size category of egg
apparatus cells, but were not expressed in the sporophytic
control tissues. Therefore, we excluded all genes with a
TPM≥3 in roots, leaves and tepals. In Table 2, we summa-
rize the ten strongest expressed *Amborella* genes enriched
in each cell size category and their best match in sequence
similarity searches. Table 2 also provides information on
three more plant species annotated for subsequent BLASTP
hits. The division of the best BLAST matches to *Amborella*
<table><caption>**Table 2** Top ten Amborella genes enriched in large (L), medium (M), and small (S) cell size categories, respectively, but with average TPM < 3 in sepals, leaves and roots. Abbreviations: AA, amino acids;</caption><thead><tr><th rowspan="2">Gene Symbol</th><th colspan="3">TPM</th><th rowspan="2">best hit*<br>(NCBI, BLASTP 2.8.1+)</th><th rowspan="2">e-value</th><th rowspan="2">Subsequent BLAST hits in other plant species</th></tr><tr><th>L</th><th>M</th><th>S</th></tr></thead><tbody><tr><td>AMTR_s00058p00172040</td><td>1811</td><td>18015</td><td>13144</td><td>Uncharacterized protein [Rosa chinensis]</td><td>5,30E-01</td><td>Gossypium barbadense, Gossypium raimondii, Quercus suber</td></tr><tr><td>AMTR_s00142p00051930</td><td>25118</td><td>10623</td><td>9569</td><td>Hypothetical protein</td><td>3,00E-72</td><td>only Amborella</td></tr><tr><td>AMTR_s00067p00156690</td><td>17713</td><td>8300</td><td>2470</td><td>Egg cell-secreted 1.4 [Amborella trichopoda]</td><td>9,00E-77</td><td>Malus domestica, Panicum miliaceum, Panicum hallii</td></tr><tr><td>AMTR_s00059p00189800</td><td>6063</td><td>3299</td><td>1124</td><td>Hypothetical protein</td><td>4,00E-74</td><td>only Amborella</td></tr><tr><td>AMTR_s01925p00009500</td><td>4982</td><td>3623</td><td>795</td><td>ACA 7, partial [Amborella trichopoda]</td><td>3,00E-49</td><td>Manihot esculenta, Jatropha curcas, Quercus suber</td></tr><tr><td>AMTR_s00074p00126160</td><td>3030</td><td>1990</td><td>572</td><td>Triacylglycerol lipase 2 [Amborella trichopoda]</td><td>8,00E-175</td><td>Elaeis guineensis, Phoenix dactylifera, Cucumis sativus</td></tr><tr><td>AMTR_s00032p00113070</td><td>1191</td><td>810</td><td>270</td><td>Hypothetical protein</td><td>6,00E-04</td><td>Cucumis sativus, Cucurbita pepo subsp. Pepo, Citrus sinensis</td></tr><tr><td>AMTR_s00010p00242830</td><td>878</td><td>626</td><td>392</td><td>CYP450 78A7 [Amborella trichopoda]</td><td>0,00E+00</td><td>Macleaya cordata, Nelumbo nucifera, Papaver somniferum</td></tr><tr><td>AMTR_s00006p00245070</td><td>788</td><td>745</td><td>178</td><td>Hypothetical protein</td><td>6,00E-70</td><td>only Amborella</td></tr><tr><td>AMTR_s00058p00172810</td><td>298</td><td>0</td><td>213</td><td>Hypothetical protein, partial</td><td>2,10E+00</td><td>only Amborella</td></tr><tr><td>AMTR_s00017p00240840</td><td>9327</td><td>12084</td><td>3892</td><td>Egg cell-secreted 1.4 [Amborella trichopoda]</td><td>9,00E-39</td><td>Papaver somniferum, Nelumbo nucifera, Citrus sinensis</td></tr><tr><td>AMTR_s00162p00083730</td><td>181</td><td>1104</td><td>1046</td><td>nsLTP [Ginkgo biloba]</td><td>8,00E-19</td><td>Corchorus capsularis, Manihot esculenta, Picea sitchensis</td></tr><tr><td>AMTR_s00040p00180260</td><td>899</td><td>1028</td><td>219</td><td>ERF3 [Amborella trichopoda]</td><td>5,00E-23</td><td>Jatropha curcas, Durio zibethinus, Medicago truncatula</td></tr><tr><td>AMTR_s04606p00004600</td><td>319</td><td>806</td><td>147</td><td>Polygalacturonase-like [Nelumbo nucifera]</td><td>5,00E-44</td><td>Nelumbo nucifera, Fragaria vesca, Hevea brasiliensis</td></tr><tr><td>AMTR_s00019p00031370</td><td>274</td><td>671</td><td>148</td><td>Polygalacturonase [Amborella trichopoda]</td><td>6,00E-110</td><td>Medicago truncatula, Trifolium pratense, Ricinus communis</td></tr><tr><td>AMTR_s00185p00055680</td><td>307</td><td>609</td><td>383</td><td>Histone H1 [Jatropha curcas]</td><td>3,00E-45</td><td>Cucurbita moschata, Populus trichocarpa, Populus euphratica</td></tr><tr><td>AMTR_s00003p00198650</td><td>250</td><td>474</td><td>89</td><td>Polygalacturonase [Elaeis guineensis]</td><td>0,00E+00</td><td>Hevea brasiliensis, Manihot esculenta, Durio zibethinus</td></tr><tr><td>AMTR_s00066p00135670</td><td>380</td><td>451</td><td>105</td><td>Polygalacturonase [Amborella trichopoda]</td><td>0,00E+00</td><td>Nelumbo nucifera, Phoenix dactylifera, Citrus sinensis</td></tr><tr><td>AMTR_s00177p00052320</td><td>281</td><td>335</td><td>91</td><td>hypothetical protein</td><td>8,00E-63</td><td>only Amborella</td></tr><tr><td>AMTR_s02876p00008560</td><td>210</td><td>222</td><td>207</td><td>hypothetical protein [Oryza sativa]</td><td>1,00E-14</td><td>Arabidopsis thaliana, Eutrema salsugineum, Morus notabillis</td></tr><tr><td>AMTR_s00003p00180860</td><td>1734</td><td>2527</td><td>5036</td><td>Hypothetical protein</td><td>2,00E-87</td><td>only Amborella</td></tr><tr><td>AMTR_s00067p00126450</td><td>75</td><td>14</td><td>980</td><td>Defensin-like protein 90 [Arabidopsis lyrata]#</td><td>4,70E-02</td><td>Zea mays, Daucus carota, Arabidopsis thaliana</td></tr><tr><td>AMTR_s00009p00257620</td><td>137</td><td>456</td><td>475</td><td>Hypothetical protein [Macleaya cordata]</td><td>1,00E-06</td><td>Citrus clementina, Theobroma cacao, Corchorus olitorius</td></tr><tr><td>AMTR_s00058p00113300</td><td>120</td><td>53</td><td>252</td><td>Lamin-like protein [Amborella trichopoda]</td><td>6,00E-60</td><td>Apostasia shenzhenica, Corchorus olitorius, Fragaria vesca</td></tr><tr><td>AMTR_s00067p00155100</td><td>7</td><td>37</td><td>240</td><td>Hypothetical protein</td><td>2,00E-40</td><td>Aegilops tauschii, Phalaenopsis equestris, Medicago truncatula</td></tr><tr><td>AMTR_s00157p00093970</td><td>19</td><td>1</td><td>227</td><td>ACA 5 [Amborella trichopoda]</td><td>2,00E-65</td><td>Phoenix dactylifera, Musa acuminata, Nelumbo nucifera</td></tr><tr><td>AMTR_s00067p00138490</td><td>63</td><td>21</td><td>183</td><td>RALF-like, hypothetical protein</td><td>5,00E-30</td><td>Elaeis guineensis, Actinidia chinensis, Arachis ipaensis</td></tr><tr><td>AMTR_s00152p00049850</td><td>35</td><td>4</td><td>156</td><td>Peroxidase 12 [Amborella trichopoda]</td><td>2,00E-123</td><td>Chenopodium quinoa, Picea abies, Spinacia oleracea</td></tr><tr><td>AMTR_s00041p00237070</td><td>103</td><td>72</td><td>155</td><td>BURP domain-containing [Amborella trichopoda]</td><td>1,00E-95</td><td>Corchorus olitorius, Sesamum indicum, Ipomoea nil</td></tr><tr><td>AMTR_s00039p00157860</td><td>0</td><td>0</td><td>145</td><td>Uncharacterized protein [Papaver somniferum]</td><td>3,00E-123</td><td>Elaeis guineensis, Macleaya cordata, Musa acuminata</td></tr></tbody></table>

ACA, alpha carbonic anhydrase; CYP450, cytochrome P450; ERF, ethylene response factor; nsLTP, non-specific lipid transfer protein; RALF, rapid alkalinization factor; TPM, transcripts per million

*Excluding the top match to the query protein if annotated as “unknown,” “uncharacterized” or “hypothetical,” except when no other significant matches were found

# tblastn search result

sequences, approximately equally between eudicots and monocots, is in agreement with *Amborella*’s phylogenetic position as a basal angiosperm, equally distantly related to these two groups (*Amborella* Genome 2013). Notably, six of the 30 genes shown in Table 2 and Supplementary Table 3 were annotated to encode “hypothetical proteins” in *A. trichopoda* and did not reveal significant sequence similarities to any protein from other flowering plant species.

More than one-third of proteins encoded by the 30 most abundantly enriched transcripts in *Amborella* egg apparatus cells were predicted to be directed to the secretory pathway, or to localize in the extracellular space. These include a potential *Amborella*-specific small secreted cysteine-rich protein (CRP) of 109 amino acids (encoded by *AMTR_s00059p00189800*) and another small secreted CRP with eight conserved cysteine residues (*AMTR_s00162p00083730*) showing sequence similarity to non-specific lipid transfer proteins (nsLTPs), which are a part of the prolamin superfamily and abundant in liverworts, mosses and all other investigated land plants (Edstam et al. 2011).

A defensin-like (DEFL) protein and a putative RALF (RAPID ALKALINIZATION FACTOR), encoded by

*AMTR_s00067p00126450* and *AMTR_s00067p00138490*, respectively, were detected in the S-group (enriched in synergid cells). RALFs are CRPs of around 5-kDa that have been identified as extracellular ligands of the *Catharanthus roseus* RLK1-like (CrRLK1L) family of receptor-like kinases, known to regulate cell expansion, pollen tube cell integrity and burst (Haruta et al. 2014; Ge et al. 2017). The *Amborella* genome was reported to encode nine RALF family proteins belonging to the major clades II, III and IV (Campbell and Turner 2017). Multiple sequence alignment of S-group-enriched clade IV-C *AMTR_s00067p00138490* with other clade IV RALFs from maize, rice and *Arabidopsis* revealed that it contains the four cysteine residues typically conserved in RALF proteins, but lacks the YISY motif conserved in clade I-III RALFs (Supplementary Figure 3). When we investigated all nine RALFs for their expression in *Amborella* sporophytic tissues and egg apparatus cells, the second clade IV-C RALF-related *AMTR_s00067p00137560* also showed weak, but selective expression in egg apparatus cells, while clade II-B (*AMTR_s00045p00202280*) and clade III-C RALFs (*AMTR_s00017p00211590*) were almost ubiquitously expressed. Strikingly, clade IV-B RALF-related
*AMTR_s00045p00207290* exhibited extremely strong expression values in the synergid-enriched group of egg apparatus cells (Fig. 4). It therefore seems likely that the respective RALF-related protein holds an important function as extracellular ligand in *Amborella* synergid cells.

The above mentioned DEFL did not reveal any similarity to synergid-expressed DEFL family proteins with known functions such as LUREs, which act as species-specific pollen tube attractants in *Torenia* and Arabidopsis (Okuda et al. 2009; Takeuchi and Higashiyama 2012), or maize ES1-4 (EMBRYO SAC1-4), which induce pollen tube tip burst (Amien et al. 2010). However, related functions cannot be excluded as DEFLs are highly polymorphic proteins, making it difficult to determine exact relationships of gene orthology (Higashiyama and Takeuchi 2015). Furthermore, other *DEFLs* with enriched expression in *Amborella* synergid cells might be present among the genes comprising egg

apparatus-expressed genes with transcripts in roots, leaves or tepals (Supplementary Table 4).

**Amborella egg cells express EGG CELL 1 and other DUF784/DUF1278 gene family members**

Importantly, we also identified four genes encoding CRPs with a prolamin-like domain (PF05617) to be specifically expressed in *Amborella* egg apparatus cells (Supplementary Table 3). These include two of the strongest expressed genes in egg cells (L-group: *AMTR_s00067p00156690* and *AMTR_s00032p00113070*), the strongest expressed gene in the M-group (*AMTR_s00017p00240840*) and one gene in the S-group (*AMTR_s00067p00155100*). A fifth CRP with prolamin-like characteristics was detected when we manually inspected the predicted protein sequence encoded by *AMTR_s00058p00172040*, which is the strongest expressed gene in egg cells (L-group) (Table 2).

**Fig. 4** Expression pattern of *Amborella* genes encoding RALFs. The classification of RALF family proteins into four major clades is according to Campbell and Turner (2017). Nine genes encoding RALF and RALF-related proteins were identified in the *Amborella* genome, belonging to clades II-B, III-B to III-D and IV-B and IV-C, respectively. Clade IV-B RALF-related *AMTR_s00045p00207290* is expressed in the control tissues (tepals, leaves, roots), but highly enriched in S-group egg apparatus cells (synergids) when compared with all other *RALF* and *RALF-related* genes. Clade IV-C RALF-related genes *AMTR_s00067p00138490* and *AMTR_s00067p00137560* are selectively present in egg apparatus cells, without detectable expression in the control tissues

<figure><img src="image_10.png" /></figure>
Prolamin-like CRPs comprise Domain of Unknown Function 784 (DUF784) and DUF1278 proteins, which are often collectively termed as ECA1 gametogenesis-related proteins (Zhang 2009). In Arabidopsis, DUF784 and DUF1278 proteins are encoded by embryo sac-specific gene families (Jones-Rhoades et al. 2007). So far, a function for prolamin-like CRPs has only been assigned for five Arabidopsis DUF1278 family members termed EGG CELL 1 (EC1). EC1 proteins are secreted by the egg cell upon sperm cell arrival and essential for successful gamete interactions during double fertilization (Sprunck et al.

2012; Rademacher and Sprunck 2013). Notably, sequence similarity searches using both *AMTR_s00067p00156690* and *AMTR_s00017p00240840* resulted in matches to proteins annotated as *Egg cell-secreted protein 1.4* (Table 2). To investigate *Amborella* prolamin-like proteins in more detail, we performed multiple sequence alignments and maximum likelihood phylogenies (Fig. 5). For sequence comparisons, we included EC1 proteins with validated expression in wheat, Arabidopsis, maize and rice egg cells (Sprunck et al. 2005; Ohnishi et al. 2011; Sprunck et al. 2012; Chen et al. 2017; Resentini et al. 2017), but also Arabidopsis DUF784

<figure><img src="image_11.png" /><figcaption>**Fig. 5** Phylogeny of EC1 and other prolamin-like proteins. Five *Amborella* prolamin-like proteins (in red) were aligned with egg cell-expressed EC1 proteins from *Arabidopsis*, wheat, maize and rice and with other DUF1278 and DUF784 family proteins from *Arabidopsis* that were shown to be expressed in synergid cells. The unrooted tree was constructed with MEGA7 (Kumar et al. 2016) using the neighbor-joining method, 1000 bootstrap replicates and Poisson correction.</figcaption></figure>

Proteins are labeled with previously published names and accession numbers, or with their respective gene identifiers. Abbreviations: ECR, EC1-related DUF1278 protein (Sprunck et al. 2014); MYB98dep, expression depends on the transcription factor Myb98 (Jones-Rhoades et al. 2007); SY, expressed in synergid cells, verified by reporter studies (Jones-Rhoades et al. 2007; Steffen et al. 2007)
and DUF1278 proteins with validated expression in synergid cells (Jones-Rhoades et al. 2007; Steffen et al. 2007). The phylogenetic tree in Fig. 5 shows individual clades formed by EC1 proteins, synergid-expressed DUF1278 proteins and DUF784 proteins, respectively. Four *Amborella* prolamin-like proteins do not locate to these clades. However, the protein encoded by *AMTR_s00017p00240840* forms a subclade with EC1 proteins from maize and rice suggesting that it represents a true *Amborella* EC1 ortholog (Fig. 5). Although a similar function for *Amborella* EC1 during double fertilization remains to be experimentally confirmed, our findings strongly suggest that the molecular mechanism of EC1-mediated sperm activation by the egg cell, as previously reported for Arabidopsis EC1 (Sprunck et al. 2012; Rademacher and Sprunck 2013), existed already in the most recent common ancestor of all extant flowering plants.

## Conclusions

We have shown that the described method for the isolation of living egg apparatus cells from the basal angiosperm *Amborella* is suitable for subsequent investigations including omics approaches, but it will also be valuable for in vitro fertilization experiments to generate zygote and embryo stages. A limited number of *Amborella* egg apparatus cells was used to generate RNA-Seq data and to identify cell type-enriched transcripts. The presented data set revealed first candidate genes for comparative evolutionary and functional studies in angiosperms, and more genes remain to be identified. It will also allow the search for molecular footprints of non-flowering seed plants, for example genes expressed in archegonia of gymnosperms. For future transcriptome studies of *Amborella* embryo sac cells, it is important to generate more biological replicates. A detailed comparison of gene expression programs in egg cells and synergid cells from *Amborella* with those from eudicot and monocot egg cells and synergid cells will contribute to the identification of evolutionary conserved molecular mechanisms involved in their specific functions during double fertilization. Future transcriptome studies should also include the *Amborella* central cell. This will require technical adaptions of the presented method to avoid the destruction of this large and highly vacuolated cell that occupies most of the volume of the embryo sac. Furthermore, the unambiguous classification of *Amborella* egg cells and synergid cells will be essential to prevent cross-contamination between pooled cell populations. Nevertheless, single-cell RNA sequencing technologies are rapidly evolving and will soon allow the profiling and classification of individual embryo sac cells, isolated from *Amborella* and other angiosperms.

**Author contribution statement** SS and TD designed the study, MFT established the cell isolation protocol and performed experiments, SP and MM processed raw RNA-seq data to enable data analyses by MFT, MM and SS. CS provided the female *Amborella* plant used for cell isolation. SS wrote the manuscript, with input from TD, MFT and CS. All authors approved the manuscript.

**Acknowledgements** We are grateful to Maximilian Weigend, Cornelia Löhne and Bernhard Reinken (Botanical Garden of the University of Bonn, Germany) for providing *Amborella* plant material. We thank Maria Lindemeier for her support in single-cell collection. Illumina deep sequencing was carried out at a genomics core facility: Center of Excellence for Fluorescent Bioanalytics (KFB, University of Regensburg, Germany). This work was supported by the ERA-CAPS Grant EVOREPRO (DR 334/12-1) to SS and TD, funded by the Deutsche Forschungsgemeinschaft (DFG).

**Open Access** This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

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