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# TRIMETHYLGUANOSINE SYNTHASE1 mutations decanalize female germline development in Arabidopsis

Lorena A. Siena<fnref n="1" /> ID, Caroline Michaud² ID, Benjamin Selles² ID, Juan Manuel Vega<fnref n="1" />, Silvina C. Pessino<fnref n="1" /> ID, Mathieu Ingouff² ID, Juan Pablo A. Ortiz<fnref n="1" /> ID and Olivier Leblanc² ID

Author for correspondence:
Olivier Leblanc

Email: olivier.leblanc@ird.fr

Received: 17 March 2023

Accepted: 14 July 2023

New Phytologist (2023) **240**: 597–612

doi: 10.1111/nph.19179

**Key words:** apospory, *Arabidopsis*, female reproductive lineage, RNA methyltransferase, TGS1.

## Summary

* Here, we report the characterization of a plant RNA methyltransferase, orthologous to yeast trimethylguanosine synthase1 (Tgs1p) and whose downregulation was associated with apomixis in *Paspalum* grasses.

* Using phylogenetic analyses and yeast complementation, we determined that land plant genomes all encode a conserved, specific TGS1 protein. Next, we studied the role of *TGS1* in female reproduction using reporter lines and loss-of-function mutants in *Arabidopsis thaliana*.

* *pAtTGS1:AtTGS1* reporters showed a dynamic expression pattern. They were highly active in the placenta and ovule primordia at emergence but, subsequently, showed weak signals in the nucellus. Although expressed throughout gametophyte development, activity became restricted to the female gamete and was also detected after fertilization during embryogenesis. TGS1 depletion altered the specification of the precursor cells that give rise to the female gametophytic generation and to the sporophyte, resulting in the formation of a functional aposporous-like lineage.

* Our results indicate that TGS1 participates in the mechanisms restricting cell fate acquisition to a single cell at critical transitions throughout the female reproductive lineage and, thus, expand our current knowledge of the mechanisms governing female reproductive fate in plants.

## Introduction

In sexual organisms, meiosis and gametic union typically pattern life cycles into alternating diploid and haploid phases (Bowman *et al.*, 2016; Pandey *et al.*, 2022). In green algae and land plants, they consist in the sporophytic and gametophytic generations, each operating specific programs for establishing the reproductive lineages. During sporogenesis, diploid sporophytic cells are specified for meiosis and form male and female haploid spores, which in turn produce multicellular gametophytes that differentiate germ cells giving rise to the gametes. Finally, the union of a male gamete (sperm) and a female gamete (egg) produces a zygote, which is a new sporophyte. Some flowering plants reproduce through strategies that sidestep meiosis and gametic fusion but retain the typical alternating sporophytic-gametophytic ontogeny (Schmidt *et al.*, 2015). These behaviors, termed gametophytic apomixis (hereafter, apomixis), perpetuate for generations the maternal genotype, an appealing outcome for crop breeding (Ozias-Akins & Conner, 2020; Khanday & Sundaresan, 2021).

Although diverse, apomictic pathways all entail, first, the formation of functional, unreduced female gametophytes (FGs) from megaspore mother cells (MMCs) after meiosis bypass

(diplospory) or from nucellar cells surrounding the MMC (apospory) and, second, the parthenogenetic development of unreduced female gametes into maternal embryos. How these two processes operating in opposite directions (i.e. sporophyte-to-gametophyte and vice-versa) are established and coordinated remains poorly understood despite recent progress in deciphering the molecular differences between sexual reproduction and apomixis (León-Martínez & Vielle-Calzada, 2019; Barcaccia *et al.*, 2020; Schmidt, 2020). Each plant generation has evolved ways to prevent the developmental programs required to establish the other one (Mosquna *et al.*, 2009; Sakakibara *et al.*, 2013) and operates a specific reproductive developmental program, that is, the formation of spores (sporogenesis) and the formation of gametes (gametogenesis; Bowman *et al.*, 2016; Böwer & Schnittger, 2021; Dierschke *et al.*, 2021). Altered expression of these programs in time and space thus seems appealing for building apomictic life cycles (Schmidt *et al.*, 2015). However, although several regulatory factors for both generations were identified in sexual models (Nakajima, 2018; Hisanaga *et al.*, 2019; Pinto *et al.*, 2019; Lora *et al.*, 2019; Hater *et al.*, 2020; Khanday & Sundaresan, 2021; Kim *et al.*, 2021; Vigneau & Borg, 2021; Y. Li *et al.*, 2021; Cai *et al.*, 2022; Huang *et al.*, 2022; Petrella

<footnote n="1">¹Instituto de Investigaciones en Ciencias Agrarias de Rosario, CONICET-Universidad Nacional de Rosario, S2125ZAA, Zavalla, Argentina; ²DIADE, Univ Montpellier, IRD, CIRAD, 34394, Montpellier, France</footnote>
et al., 2022; Sanchez-Vera et al., 2022), no clear hookup with
natural apomictic systems has emerged yet, except evidence for
members of the RNA-directed DNA methylation pathway (Gri-
manelli, 2012; Selva et al., 2017; Galla et al., 2019). On the other
hand, and contrary to inheritance studies suggesting a limited
number of loci controlling apomixis, variation in the expression
of large numbers of genes affecting a wide range of cellular activ-
ities usually differentiates sexual and apomictic developments
(Brukhin & Baskar, 2019; Schmidt, 2020). Difficulties in acces-
sing the cell types of interest have certainly hindered the deci-
phering of these changes and relevant orchestrating factors
remain elusive thus far, except for a few candidates that remain
functionally uncharacterized, for example, APOLLO (Corral
et al., 2013) and QUI-GON JINN (Mancini et al., 2018). More-
over, deletion mapping in *Hieracium* apomicts identified two
loci, LOSS OF APOMEIOSIS and LOSS OF PARTHENO-
GENESIS (Koltunow et al., 2011), whose action remains unre-
solved at the molecular level. Instead, comparative genomics in
aposporous *Pennisetum squamulatum* and deletion mapping
in diplosporous *Taraxacum officinale* identified two candidates
encoding, respectively, a BABY BOOM-LIKE (BBML) tran-
scription factor of the APETALA2 family and the PARTHENO-
GENESIS (PAR) transcription factor (Conner et al., 2015;
Underwood et al., 2022). Heterologous expression of these pro-
teins in sexual relatives promotes the formation of parthenoge-
netic embryos that can give rise to viable maternal progenies
(*P*BBML and *Oryza sativa* BBM homologs) and divisions in egg
cells (*To*PAR), at least in species from their respective clades
(Conner et al., 2017; Khanday et al., 2019; Underwood
et al., 2022).

In *Paspalum notatum* apomicts, we recently identified a
candidate gene encoding a RNA methyltransferase with a
S-adenosyl-L-methionine (SAM; pfam09445) domain typical of
trimethylguanosine synthase1 (*TGS1*) in yeast (*Saccharomyces
cerevisiae*) and animals (Siena *et al.*, 2014; Fig. 1a). In these organ-
isms, transcription by RNA POLYMERASE II generates RNAs
bearing a 7-methylguanosine (m⁷G) 5'-end cap, whose conversion
into m²,²,⁷G-cap by TGS1 is essential for biogenesis and trafficking
of several types of RNA molecules, including small nuclear and
nucleolar RNAs (Mouaikel *et al.*, 2002; Hausmann *et al.*, 2008;
Verheggen & Bertrand, 2012; Cheng *et al.*, 2020); quiescence-
induced miRNAs (Martinez *et al.*, 2017); human telomerase RNAs

(Chen et al., 2020); heterochromatic noncoding RNAs involved in
the establishment of centromeric heterochromatin (Yu et al., 2021);
and selenoprotein mRNAs (Wurth et al., 2014). While Tgs1p in
yeast consists in a SAM domain and a short N-tail harboring a
nuclear localization sequence (NLS) region, animal TGS1 proteins
have extended N-termini (Fig. 1a) embracing transcriptional regu-
lation and other functions beyond RNA processing (Zhu
et al., 2001; Jia et al., 2012). TGS1 mutations usually produce mild
phenotypes related to mitosis and growth, including low tempera-
ture sensitivity in growing yeast (Mouaikel et al., 2002); pericentro-
meric heterochromatin defects in S. pombe (Yu et al., 2021); and
reduced cell growth in mammals (Lemm et al., 2006). In contrast,
they compromise reproduction in budding yeast (no meiosis com-
mitment; Qiu et al., 2011), *Drosophila* (germline and pupal altered
development; Komonyi et al., 2005; Cheng et al., 2020), and mice
(embryogenesis defects; Jia et al., 2012).

In contrast to animals and yeast, little information is currently
available for TGS1 function and contribution to plant develop-
mental biology. Previous reports have shown a role in chilling
resistance in *Arabidopsis thaliana* (Gao et al., 2017) and flowering
time in *Lupinus angustifolia* (Taylor et al., 2021). In addition,
*TGS1* expression levels and rates of residual sexuality in *P. notatum* apomicts were found to correlate positively (Siena et al., 2014), an observation consistent with aposporous-like FGs
formation in sexual *P. notatum* transformants expressing an anti-
sense RNA-*PnTGS1* construct (Colono et al., 2019). To gain
further knowledge into the functional characterization of TGS1
proteins during plant reproduction, we first generated phyloge-
netic trees from plant TGS1 sequences and provide evidence for
the conservation of a specific domain architecture across land
plants. Next, by using reporter imaging and reverse genetics in *A. thaliana*, we demonstrated that the conserved TGS1 isoform is
required for female germ cell specification and that loss-of-
function causes phenotypes reminiscent of aposporous apomixis.

**Materials and Methods**

Plant materials

*Arabidopsis thaliana* (L.) Heynh seeds were stratified in the dark at 4°C for 2 d and then transferred to a growth chamber (22°C, long-day conditions). All crosses were performed using

**Fig. 1** Characterization of plant TGS1 proteins. (a) Scaled drawings of representative plant TGS1 methyltransferases. Note the unusual intragenic duplication of the SAM domain in AtTGS1-LIKE. HR, hydrophobic residues. At, *Arabidopsis thaliana*; Hs, *Homo sapiens*; Pn, *Paspalum notatum*. (b) Com- plementation in yeast *Atgs1* mutants expressing full-length and truncated PnTGS1 proteins growing at low (growth-sensitive) and high (growth- insensitive) temperatures. (+), wild-type (WT) strain BY4741. (c) Phylogenetic tree of Tgs1 orthologs in green lineages. Colored triangles show TGS1-LIKE monophyletic clades. Black and white dots indicate moderate branch support by the aBayes criterion: 0.5–0.75, and 0.75–0.9, respectively. All other nodes have values > 0.95. Black and gray: green algae encoding TGS1 proteins possessing and lacking a WW domain, respectively; blue, Bryophytes; pink, ferns; bronze, gymnosperms; green, angiosperms; red, land plant clades lacking TGS1-LIKE. (d) RT-PCR for AtTGS1 and the ACTIN11 control gene in *Arabidopsis* tissues. (e–j) GUS staining patterns in *pAtTGS1:AtTGS1-GUS* reporter lines showing accumulation in (e) placenta and ovule primordium (arrowheads); (f) young ovules (arrowheads) before MMC differentiation; (g) an ovule containing a MMC (arrowhead); (h) an ovule containing a mature MMC (arrow- head); and (i, j) postmeiosis ovules containing a surviving megaspore (arrowhead) and three degenerated spores (arrows). (k–q) Confocal images of *pAtTGS1:AtTGS1-GFP* reporter lines showing fluorescent signals in a four- (k) and an eight-nucleated (l) female gametophyte; a mature gametophyte (m); a zygote (n); a two-celled embryo (o); a 3-d after pollination (DAP) isolated globular embryo (p); and mature male gametophytes (q). Note the strong sig- nal in the egg cell (l, m). cc, central cell; ec, egg cell; em, embryo; en, endosperm; fm, functional megaspore; pn, polar nuclei; zy, zygote. Bars: (g, h, q) 10 µm; (e, f, l–p) 30 µm.
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emasculated buds that we hand-pollinated 2 d after emasculation, unless stated otherwise. Ecotype Columbia (Col-0) was used as wild-type (WT) plants. T-DNA insertion lines for *AtTGS1* (*At1g45231*) were *tgs1-4* (SALK_020980) and *tgs1-5* (SALK_071682; see Supporting Information Fig. S1 for mutants’

selection). Fluorescent reporter lines were used for female germline precursors (*pKNU:nlsYFP*; Payne *et al.*, 2004); synergid cells (*pNTA:nTdTomato*; Kong *et al.*, 2015); FG cell types using *FGR7.0* (Völz *et al.*, 2013) that contains *EC1:NLS_3xdsRed* (egg cell), *DD22:NLS_YFP* (central cell/endosperm), and *DD2*:
NLS_3xGFP (synergid cells/endosperm); gametophytic cell fate and ploidy (pWOX2-CENH3-GFP; Keçeli *et al.*, 2017); and early embryo cell fate (pS4:nGFP; Kong *et al.*, 2015) and patterning (pDRN: GFP; Cole *et al.*, 2009). *tgs1* mutants containing these reporters were obtained from F2 plants, derived from crosses between *tgs1* mutants and the reporter lines, PCR genotyped to recover *tgs1* homozygous plants, and finally selected based on non-segregating expression pattern of the reporter in ovules (*n* > 50). Observations reported here were performed using derivatives of one selected F₂ line per combination. For *pAtTGS1:AtTGS1-GFP* and *pAtTGS1:AtTGS1-GUS* reporter lines, we transformed Col-0 plants by floral dipping using binary vectors (see 'Molecular procedures' in the Materials and Methods section). T1 seeds were grown on solid Murashige & Skoog (MS) medium supplemented with BASTA (10 mg l⁻¹), hygromycin (30 mg l⁻¹), or kanamycin (50–200 mg l⁻¹) as selective markers. Germination tests were performed using two batches of 100 sterilized seeds per genotype that were sown onto germination medium (half MS salts supplemented with 1% agar) in Petri dishes and monitored for 10 d under a stereomicroscope. Developmental alterations in plantlets were screened under a stereomicroscope.

## Phylogenetic reconstruction analyses

Sequences of green algae (n=12) and land plants (n=59) species were searched in public databases by BLASTp (eventually tBLASTn) using Tgs1p (Saccharomyces Genome Database ID: SGD:S000006078; www.yeastgenome.org/) as a query. Amino acid sequences (Table S1) were first screened for known domains (e.g. SAM, WW, and NLS) using INTERPRO (https://www.ebi.ac.uk/interpro/search/sequence/) and MOTIFSCAN (https://myhits.isb-sib.ch/cgi-bin/motif_scan) and searched for conserved residues using the GenomeNet Bioinformatics Tools (https://www.genome.jp/tools/motif/MOTIF2.html). Phylogenetic analyses were performed using the Next Generation Phylogeny web service (https://ngphylogeny.fr) with the following workflow: MAFFT for multiple amino acid sequence alignments using a Blosum62 matrix and default parameters for gap extension and opening penalties; BMGE for alignment curation and selection of informative blocks using a Blosum62 matrix, a minimum block size of 5 and the default for remaining parameters; PhyML for tree inference (Blosum62 matrix; BEST option for tree topology search; aBayes criterion for branch support); and Newick default display for tree rendering.

## Yeast procedures

Wild-type (BY4741) and Δ*tgs1* strains were transformed following the LiAc method (Gietz & Schiestl, 2007) using plasmids encoding full-length or truncated TGS1 proteins from *Paspalum notatum* Flüggé (see 'Molecular procedures' in the Materials and Methods section). Yeast cell lines were grown in liquid YPDA medium containing auxotrophic selecting markers (0.2% Ura, 2% Met and 1% Leu) for 24 h at 30°C under shaking. For functional complementation, cells were harvested by

centrifugation (1200 g, 5 min), resuspended into sterile water to OD₆₀₀ = 0.5, and sequentially diluted 10, 100 and 1000 times in sterile water. Ten µl of each dilution was spotted onto agar medium supplemented with 0.5% glucose and the auxotrophic selecting markers and incubated 4 d at 30°C or 9 d at 18°C before scoring. Yeast strains, BY4741 (MATa; ura3Δ0; leu2Δ0; his3Δ1; met15Δ0) and Δ*tgs1* (BY4741; MATa; ura3Δ0; leu2Δ0; his3Δ1; met15Δ0; YPL157w::kanMX4), were obtained at http://www.euroscarf.de.

## Molecular procedures

For T-DNA insertion line genotyping, genomic DNA extracted from leaf tissue was assayed by PCR. For RT-PCR analyses, RNA was isolated using TRIzOL (Invitrogen) and treated with DNaseI. Leaves collected at three developmental stages (plantlets, rosette, and flowering onset) and floral buds collected from presporogenesis to 2 d after pollination were used as samples for vegetative and reproductive tissues, respectively (three WT and *tgs1* plants/sample). Following reverse transcription using 1 µg of total RNA and Invitrogen SuperScript III Reverse Transcriptase according to manufacturer's instructions, cDNAs were amplified for *AtTGS1* and *ACTIN11* as a control for constitutive expression. To generate constructs reporting *AtTGS1* expression, we ordered from GenScript USA Inc. a pUC57-KAN vector containing a synthesized DNA fragment spanning 1732 base pairs (bp) upstream of the initiation codon up to the last amino acid before the stop codon of *AtTGS1* (4912 bp) and flanked by the attL Gateway sites. This vector was used as an entry clone in LR reactions (Invitrogen) with two Gateway destination vectors, pGWB604 and pGWB433, to produce *pAtTGS1:AtTGS1-GFP* and *pAtTGS1:AtTGS1-GUS*, respectively. For yeast expression vectors, coding sequences were amplified from cDNA with specific primers and directionally cloned into the pENTR/D-TOPO entry vector (Invitrogen). Entry plasmids were recombinased (LR clonase II, Invitrogen) with destination vectors pRS423-GPD-ccdb-EGFP (AddGene #14198) and pRS426-GPD-ccdb-DsRed (AddGene #14372) for monitoring the expression of PnTGS1 and Tgs1p, respectively. All primers are shown in Table S2.

## Whole-mount preparations and microscopy

Pollen grains were collected from open flowers, placed under a coverslip in sterile water containing 1 µg ml⁻¹ DAPI (4',6-diamidino-2-phenylindole) for 5 min. Ovule clearing and histochemical detection of the *uidA* reporter gene product were performed as described previously (Autran *et al.*, 2011). Callose detection in ovules stained with aniline blue was performed as described previously (Cao *et al.*, 2018). Observations were made on either a Zeiss Axio Imager or a Leica DM2500 under Differential Interference Contrast (DIC) optics for cleared ovules. Fluorescence in ovules mounted in 30% glycerol was imaged on a Zeiss Axio Imager using a 450–490 nm excitation filter and a BP 525/
50 nm bandpass filter. For *pAtTGS1:AtTGS1-GFP* and *pWOX2-CenH3-GFP* signals, imaging was also performed using a confocal microscope (Leica TCS SP8 and Zeiss LSM880 Airyscan).

Flow cytometry

Cell ploidy in bulks containing four mature seeds of WT or *tgs1*
plants was determined by estimating nuclear DNA content using a
flow cytometry procedure adapted from Matzk *et al.* (2000). Seeds
were placed in a glass Petri dish into a 50-µl drop of extraction
buffer (Tween20 0.5%, citric acid 0.1 M) and grinded using a glass
pestle. After collecting the remains by rinsing the pestle with
150 µl of sodium phosphate monobasic 0.4 M, the extracts were
filtered (30-µm mesh) and the nuclei solution was supplemented
with 4 µl of Ribonuclease A (0.5 mg ml⁻¹). After incubation at
room temperature for 5–10 min, 10 µl of 1 mg ml⁻¹ Propidium
iodide (Sigma, P4170) was added to the solution and DNA con-
tents were measured using a Becton-Dickinson FACSAria™ II
workstation with excitation and emission wavelengths of 538 and
617 nm, respectively. We used FLOWJO v.10.0.7 (BD Biosciences,
Wokingham, UK) for data analysis (at least 2500 nuclei/scoring
window, except for samples with low nuclei concentration) and
graphical views. The null hypothesis that the proportions of nuclei
corresponding to three DNA content classes (2×, 3×, >3×)
obtained from each *tgs1* bulk were similar to that estimated from
17 bulks of WT seed was tested using Pearson's chi-squared tests.
When rejected, significant increase in 3× nuclei compared with
2× nuclei was assessed using chi-squared tests.

Results

TGS1 plant sequences and phylogenetic relationships

Previous works showed that a single gene encodes TGS1 in all
eukaryotes, but *Arabidopsis* and rice possess two copies (Fig. 1a;
Mouaikel *et al.*, 2002, 2003). Since *TGS1* family members have
remained largely uncharacterized in the green lineage, we first
showed by successful complementation in Δ*tgs1* yeast that the pro-
duct encoded by the *Paspalum TGS1* candidate gene for apomixis
is a functional homolog of Tgs1p (Fig. 1b). Next, we performed
phylogenetic analyses using protein sequences retrieved by BLASTP
searches from sequenced genomes of Chlorophytes (green algae;
*n* = 12) and Streptophytes, including 57 land plant species and
two of their Charophytes (freshwater algae) ancestor species
(Table S1). As suggested in *Arabidopsis* (Gao *et al.*, 2017),
sequences alignment first revealed highly conserved C-termini har-
boring a nuclear localization sequence (NLS) adjacent to the SAM
domain, while N-tails varied in length and domain composition
depending on the presence of a protein-interacting WW domain
(Sudol, 1996) and two stretches of conserved residues (> 75%
identity) with a hydrophobic pattern (LMKEMNDLGLPVSF and
AIRALGHLFRLKEVFLEDD for *A. thaliana*; hydrophobic resi-
dues are underlined; Figs 1a, S2; Notes S1). BLASTP searches using
the consensus sequence of these hydrophobic patterns produced
no hit outside of the Viridiplantae. Although their function
remains speculative, hydrophobic residues participate in

stabilization of protein structure and complexes. Interestingly, and
contrary to the WW domain that is dispensable for complement-
ing Δ*tgs1* yeast strains (Gao *et al.*, 2017), our data suggest that
complementation in Δ*tgs1* yeast requires at least one of these two
motifs, as N-terminally truncated PnTGS1 containing the WW
domain, the NLS and the SAM domain were unable to rescue
Δ*tgs1* strains (Fig. 1b).

Phylogenetic reconstruction using conserved blocks from
alignments recapitulated green plants phylogenomics (One
Thousand Plant Transcriptomes Initiative, 2019; Figs 1c, S3;
Notes S2). Unsurprisingly considering the many questions per-
sisting on their relationships and evolution, we obtained a poor
resolution among the Chlorophytes algae sequences used here.
Nevertheless, all species possess a single gene encoding a protein
that contains a potentially catalytically active SAM domain, but
highly variable N-tails regarding length and conserved motifs
composition. In contrast, phylogenetic resolution in Strepto-
phytes first identified a TGS1 ortholog in land plants, whose core
components (hydrophobic residues; WW, NLS, and SAM
domains; Fig. 1a) were conserved since they diverged from the
Charophytes lineage. Furthermore, three monophyletic clades,
clustering paralogs with large N-tail truncations lacking the WW
domain, can be distinguished at different evolutionary times,
indicative of distinct ancient duplication events followed by sig-
nificative structural rearrangements in ancestors of ferns, Gym-
nosperms and Angiosperms. Accordingly, we followed Gao
*et al.* (2017) nomenclature adopting *TGS1* for the genes encod-
ing the conserved WW-SAM protein and *TGS1-LIKE* for the
paralogous genes. Finally, sporadic duplications eventually fol-
lowed by rearrangements (e.g. incomplete intragenic duplication
in *A. thaliana* *TGS1-LIKE*; *TGS1* duplication in *Glycine max* fol-
lowed by N-tail truncation) and frequent losses of acquired para-
logs in extant clades (families or species) of phylogenetic trees
complete TGS1s evolutionary trajectory in plants (Figs 1a, S3).
While TGS1-LIKE paralogs appear dispensable, the retention of
a single, highly conserved architecture in TGS1 proteins suggests
that they fulfill essential functions in plants.

TGS1 expression during female reproductive development

Our previous analyses of *TGS1* expression and depletion in *P. notatum* ovules (Siena *et al.*, 2014; Colono *et al.*, 2019) and the phylogeny reconstitution previously reported prompted us to further decipher the function of TGS1 using the plant model *A. thaliana*. First, RT-PCR experiments revealed higher *TGS1* transcripts accumulation in reproductive organs compared with vegetative tissues (Fig. 1d). Next, to precisely monitor protein accumulation, we generated *pAtTGS1:AtTGS1-GUS/GFP* reporter lines to express C-terminal translational fusions of TGS1 to the beta-glucuronidase or the GFP protein, respectively. Reporter expression analyses for both constructs (at least three independent transformants each and *n* > 100 ovules per stage) agreed with RT-PCR experiments (Figs 1e–q, S4). Signal accumulated to high amounts in cells of the placenta, the sporophytic tissue that differentiates the ovules, and in those of emerging ovule primordia (Figs 1e, S4a). As ovules grew, staining intensity decreased along the proximal-distal axis and,
before meiosis initiated, the L1 cell layer and the nucellus, including the MMC, showed a weak, patchy expression (Figs 1f–h, S4b). These two contrasting expression domains persisted after meiosis but, in contrast to the MMC and the degenerating spores of the tetrad, the surviving functional megaspore (FM) stained intensely and was embedded in the proximal AtTGS1 expression domain (Fig. 1i). This pattern in subcellular localization might also indicate differences in activity as reported in yeast and humans, where Tgs1 can exist as two species with different substrate and localization specificities (Verheggen & Bertrand, 2012). The FM then produces through three mitoses a coenocyte that differentiates into a mature, seven-celled female gametophyte (FG) typically containing: three antipodal cells; two synergid cells (SCs); and two gametes, the haploid egg cell and the dihaploid central cell (Hater et al., 2020). We observed diffuse, fading into background signals within developing coenocytic gametophytes and surrounding maternal tissues (Figs 1j, k, S4c). During nuclear migration, but before cellularization and differentiation occurred within FGs, the signal increased in the precursor nucleus of the egg cell (Fig. 1l) and, at maturity, it became restricted to gametic cells, being more intense in the egg (Fig. 1m). Mature male gametophytes showed a similar pattern, with diffuse signals in the generative cell and bright intensities in sperm cells (Fig. 1q). Finally, during early seed development (1–3 d after fertilization), signal accumulated essentially in the zygote (Figs 1n, S4d,e) and the developing embryo (Figs 1o,p, S4f–j) regardless of reporter inheritance, therefore suggesting early activation of the paternal allele (Fig. S4d–j). Note that no signal was detected throughout endosperm development, except for faint staining at early developmental stages (up to three rounds of nuclear divisions; Fig. 1n), suggesting maternal carryover from the central cell rather than transcriptional activity.

## TGS1 restricts female germline specification

The peculiar domain organization of TGS1 in plants and its expression pattern in developing ovules suggest a preponderant role during plant reproduction. To test this hypothesis, we further characterized two *Arabidopsis* T-DNA insertion lines

carrying *tgs1* loss-of-function alleles, SALK_020980 (*tgs1-4*) and SALK_071682 (*tgs1-5*; see Fig. S1a,b for details). High proportions of aborted seeds in *tgs1* heterozygous and homozygous plants (Fig. S1c,e) and segregation against *tgs1* mutants in progenies of heterozygous plants (Table 1) suggested defects in gametogenesis or embryogenesis. Indeed, reciprocal crosses between WT and *tgs1l+* plants revealed reduced maternal transmission of *tgs1* alleles (Table 1). Finally, in crosses between *tgs1-4* mutants heterozygous for *pAtTGS1:AtTGS1-GUS* and WT plants, gametes carrying the GUS construct were significantly more transmitted (χ² (1, n=89) = 4.05, P<0.05; Table 1) as observed for the WT allele in progenies derived from *tgs1l+* x WT crosses. This suggested that our constructs are fully functional.

Since *tgs1* pollen development showed no visible defects when stained using DAPI (>95% of tricellular pollen grains in three individuals for each mutant line; Fig. S1d), we investigated ovules using differential interference contrast (DIC) microscopy and fluorescent protein reporters (Fig. 2). Before meiosis, most WT ovules (95%, n=400) differentiated a single MMC (Fig. 2a) while the remaining ones had two, sporadically three, cells resembling MMC, that is, also exhibiting enlarged size and nucleus compared with surrounding cells. In contrast, *tgs1-4* and *tgs1-5* ovules exhibited one extra enlarged cell at significantly higher proportions than WT ovules (two-tailed *P* value for Fisher's exact test <0.0001; Fig. 2c,d), respectively: 16% (n=183) and, 18% (n=150). Furthermore, *tgs1-4* ovules exhibited two extra enlarged cells at a frequency of 2% (n=183; Fig. 2d). To explore whether the enlarged cells had MMC identity, we analyzed *tgs1-4* plants expressing *pKNU:nlsYFP*, a preferential marker of MMCs also detected in megaspores of WT ovules (Payne et al., 2004; Fig. 2b,f,h). In *tgs1-4* ovules containing multiple enlarged cells, we found that *pKNU* was active in only one of them, suggesting only one is specified for meiosis (Fig. 2e,g).

Next, observations during meiosis revealed that WT (n=95) and mutant (n=109) ovules contained the meiotic products of a single tetrad (Fig. 2h–n). *pKNU:nlsYFP* signal was restricted to the chalazal spore in most WT ovules (n=93; Fig. 2h) while 12 *tgs1-4* degenerating tetrads (11%) had two fluorescent chalazal spores (Fig. 2i). Callose is a typical marker of meiosis, which

<table><caption>**Table 1** Transmission of *tgs1* alleles in *Arabidopsis thaliana*.</caption><thead><tr><th rowspan="2">Crosses</th><th colspan="3">Progeny genotypes</th><th rowspan="2">Expected ratio</th><th rowspan="2">χ<sup>2</sup></th></tr><tr><th>Tgs1/Tgs1</th><th>Tgs1/tgs1</th><th>tgs1/tgs1</th></tr></thead><tbody><tr><td>tgs1-4/Tgs1 × tgs1-4/Tgs1</td><td>73</td><td>77</td><td>33</td><td>1:2:1</td><td>22.07***</td></tr><tr><td>tgs1-5/Tgs1 × tgs1-5/Tgs1</td><td>45</td><td>47</td><td>20</td><td>1:2:1</td><td>17.03***</td></tr><tr><td>tgs1-4/Tgs1 × Col-0</td><td>106</td><td>52</td><td>—</td><td>1:1</td><td>18.45***</td></tr><tr><td>Col-0 × tgs1-4/Tgs1</td><td>97</td><td>111</td><td>—</td><td>1:1</td><td>0.94<sup>ns</sup></td></tr><tr><td>tgs1-5/Tgs1 × Col-0</td><td>77</td><td>39</td><td>—</td><td>1:1</td><td>12.44***</td></tr><tr><td>Col-0 × tgs1-5/Tgs1</td><td>32</td><td>30</td><td>—</td><td>1:1</td><td>0.06<sup>ns</sup></td></tr></tbody><thead><tr><th></th><th>Tgs1/tgs1-4<sup>GUS</sup></th><th>Tgs1/tgs1-4</th><th></th><th></th><th></th></tr></thead><tbody><tr><td>tgs1-4/tgs1-4:GUS/- × Col-0</td><td>54</td><td>35</td><td>—</td><td>1:1</td><td>4.05**</td></tr><tr><td>tgs1-4/tgs1-4:GUS/-</td><td>44</td><td>48</td><td>—</td><td>1:1</td><td>0.10<sup>ns</sup></td></tr></tbody></table>

Col-0 was used as wild-type (WT). χ², statistic value of two-sided χ² tests indicating nonsignificant (ns) and significant deviation from the expected ratio (**, P < 0.05; ***, P < 0.001). GUS: *pAtTGS1:AtTGS1-GUS*; *tgs1-4GUS*: complementation of *tgs1-4* by GUS.
<figure><img src="image_7.png" /><figcaption>**Fig. 2** TGS1 depletion triggers additional female germline in *Arabidopsis thaliana*. Ovule development in wild-type (WT) and *tgs1* plants carrying *pKNU:nlsYFP* (a–n) and *pWOX2-CENH3-GFP* (o–u). (a–g) Premeiotic ovules containing (a–e) enlarging MMCs (arrowhead) and (f, g) mature MMCs (arrowhead) accompanied by MMC-like enlarged cells (asterisks) in *tgs1* ovules. (h–u) Early steps of gametophytic development in WT ovules showing a single tetrad with one surviving megaspore (h, j, o) and a binucleated FG (q). Note the reminiscent signal of *pKNU:nlsYFP* and the strong dotted signal of *pWOX2-CENH3-GFP* indicating reduced FM. In contrast, *tgs1* ovules at meiosis showed several phenotypes, including tetrads with two FM (i, k, p) resulting in multiple reduced, binucleated FGs (r); *pKNU:nlsYFP* and *pWOX2-CENH3-GFP* ectopic signal in enlarged MMC-like cells near the FM (l, t) resulting in the development of unreduced binucleated FGs (m, n, u); and extremely enlarged MMC-like cells lacking *pWOX2-CENH3-GFP* signal near a correctly specified, reduced FM (s). Note that (m, n) show different optical plans of the same ovule (see also Supporting Information Fig. S5), containing a binucleate FG (m) and a tetrad of megaspores (n). Fluorescence signals detected for reporters and callose were merged with DIC images in (b, e–i, l, n–u) and (j, k), respectively. Arrowheads indicate the legitimate female germline cells initiating megasporogenesis (premeiotic ovules) and gametophyte development, while asterix indicates candidate precursors for additional female germline development. Bars, 10 µm.</figcaption></figure>

starts depositing in MMC cell walls and shows high accumulation in dyads and tetrads, while it quickly disappears around the surviving FM (Webb & Gunning, 1990). Interestingly, callose patterns revealed by aniline blue staining showed no deposition in the walls between the two chalazal spores (7/50 tetrads in *tgs1* ovules; Fig. 2k). Note that this pattern was not detected in WT tetrads where callose typically deposited around the three micro-pylar spores (Fig. 2j).

At later stages of meiosis, in ovules containing extra MMC-like cells, *pKNU:nlsYFP* signal appeared fading in the surviving megaspores of the tetrad while strong expression was detected in the enlarged nucleus of most MMC-like cells (n=7 out of 8 ovules; Fig. 2l). Finally, in five ovules, we could observe a binucleated embryo sac lacking KNU expression and adjacent to the legitimate tetrad (Figs 2m,n, S5). Therefore, while meiosis occurs in the specified MMC, MMC-like cells in TGS1-deficient ovules retain a potential for germline specification that expresses during the late stages of meiosis. These results suggest that KNU repression by TGS1 in nucellar cells surrounding the MMC contributes to confining female germline specification to a single cell.

## TGS1 loss-of-function relaxes control of gametophytic and gametic developments

Adoption of gametophytic program was assessed using the *pWOX2-CENH3-GFP* reporter (Keçeli *et al.*, 2017). While only FM and FG nuclei were labeled in WT ovules throughout gametophytic development (Figs 2o,q, 3d), we detected signals in all additional surviving spores of *tgs1* tetrads (n=26; Figs 2p, S6a) and in all but a few MMC-like cells adjacent to a FM (n=5; Fig. 2t) among 105 ovules. These observations indicate that *tgs1* mutations can promote ectopic gametophyte formation in both somatic nucellar cells and tetrad meiotic products, therefore suggesting that in *Arabidopsis* TGS1 acts to restrict the number of cells committed to form a FG both within and outside of the canonical lineage. In support, we observed in ovules at early gametogenesis (n=86): a single reduced, binucleated FG along with one additional surviving spore (n=10; Fig. S6d); two reduced, binucleated FGs (n=5; Figs 2r, S6g); and one unreduced binucleated FG (n=2; Figs 2u, S6e), indicating that both types of spores (i.e. reduced and unreduced) are likely functional.
<figure><img src="image_8.png" /><figcaption>**Fig. 3** TGS1 depletion causes multiple gametophytic and gametic developments in *Arabidopsis thaliana*. DIC observations (a–c) and signals from fluorescent reporters (d–j) in WT and *tgs1* ovules at anthesis. Wild-type (WT) ovules (a, d, f, i) typically contain one eight-nucleated, seven-celled FG composed of three antipodal cells (an) at the chalazal end; a central cell (cc) which polar nuclei (pn) have fused; and one egg cell (ec) and two synergid cells (sy) at the micropylar end. Most common defects observed in mutant ovules were: (b) multiple FGs; abnormal number of nuclei in the central region (e) and the micopylar region (g); alterations in synergid cells position (c, h, j); and mixed identity in the egg apparatus (g, h; see also Supporting Information Fig. S7). Confocal projections in (d, e) were obtained from *pWOX2-CNH3-GFP* ovules and epi-fluorescence signals in (f–h) and (i, j) from *FGR7.0* (ec: *EC1::NLS_3xdsRed*; *sy: DD2::NLS_3xGFP*; *cc: DD22::NLS_YFP*) and *pNTA:nTdtomato* ovules, respectively. Bars, 10 µm.</figcaption></figure>

Finally, our observations support the hypothesis that extra MMC ectopically express both *pKNU:nlsYFP* and *pWOX2-CENH3-GFP*.

At later stages of gametogenesis, while WT mature ovules always contained a typical seven-celled FG (*n* > 200), we observed in *tgs1-4* and *tgs1-5* cleared ovules (*n* = 116 and *n* = 100, respectively) multiple FGs (6.5%) as well as FGs affected in nuclei number or positioning (15%; Fig. 3a–c). These phenotypes were further characterized in *tgs1* lines carrying *pWOX2-CENH3-GFP*, which revealed mature FGs with extra nuclei (Fig. 3d,e), and fluorescent reporters for gametic (*FGR7.0*) and SC fates (*FRG7.0*; *pNTA:nTdtomato*). Using *FGR7.0* (*n* = 111), gametic cells usually exhibited the WT phenotype (Fig. 3f) whereas SCs showed doubling, likely after an extra division (2.3%; Figs 3g, S7a,b); altered positioning (9.2%; Fig. 3h, S7c,d); and egg cell marker ectopic expression (31.1%; Fig. S7a,b). Such alterations were never observed in WT FGs (*n* > 200). The gametic potential in SCs in *tgs1* was confirmed by observations using *pNTA:nTdtomato* in *tgs1* FGs (Fig. 3i,j). Furthermore, cell fate determination in the egg apparatus of *tgs1* FGs seemed uncoupled from nucleus positioning since identity was maintained in SCs found at abnormal positions and, reciprocally, acquisition of egg cell identity occurred in correctly positioned

SCs (Figs 3, S7). Nevertheless, most FGs exhibited at least one properly specified SC according to *FRG7.0* and *pNTA* expression at the micropylar end, therefore suggesting that *tgs1* FG can mediate pollen tube attraction and sperm delivery.

### *tgs1* seeds show defects in both fertilization products

Examination of fertilized *tgs1* ovules in plants revealed defects in the endosperm, the embryo or both. First, we observed seeds 2–4 d after pollination (DAP) lacking endosperm or containing an abnormal endosperm with few enlarged nuclei (15 and 5 out of 161 *tgs1-4* seeds and 6 and 4 out of 167 *tgs1-5* seeds, respectively; Fig. 4a–e). Cell fate reporter analysis in 1–3 DAP *tgs1* seeds further revealed abnormal identity in endosperm nuclei as ectopic signal for embryo-specific *pS4:nGFP* reporter was detected (12/58 and 16/136 in *tgs1-4* and *tgs1-5* ovules, respectively; Fig. 4f–m), while both endosperm reporters in *FGR7.0* (i.e. *DD22::NLS_YFP* and *DD2::NLS_3xGFP*) showed the expected signal in endosperm nuclei of most ovules (86/91 and 31/33 in *tgs1-4* and *tgs1-5* ovules, respectively; Fig. 4n–q). Interestingly, ectopic expression of *FGR7.0* endosperm reporters was detected in *tgs1* embryos at 2 DAP (15/39 and 8/40 in *tgs1-4* and *tgs1-5* ovules, respectively; Fig. 4o–q). Therefore, the ectopic signals generated
<figure><img src="image_9.png" /><figcaption>**Fig. 4** Developmental effects in *tgs1* seeds of *Arabidopsis thaliana*. (a) Wild-type (WT) ovule showing normal endosperm development at 2 d after pollination (DAP). (b, c) DIC image (b) and *pDRN:GFP* expression (c) in an aborted *tgs1-4* seed containing a wt, globular embryo and lacking endosperm. (d, e) Two different optical plans of the same 2-DAP seed containing a globular embryo (d) and abnormal endosperm nuclei (arrowheads). (f–m) Specific expression of *pS4:nGFP* in a zygote (f) and developing embryos (g–i) in WT seed compared with *pS4:nGFP* ectopic expression in endosperm nuclei (arrowheads) of *tgs1-5* seeds (j–m). The embryo is indicated by an asterisk. (n) WT seed showing specific expression of *pEC1:NLS_3xdsRed* (magenta) in a two-celled embryo (arrowhead) and of *DD2:NLS_3xGFP* (green) in endosperm nuclei. (o–q) *FGR7.0/tgs1-4* seed with an embryo (arrowhead) exhibiting mixed identities (o, merged signals; p, *pEC1:NLS_3xdsRed* signal; q, *DD2:NLS_3xGFP*). Bars: (b, c) 10 µm; (f–q) 30 µm; (a, d, e) 50 µm.</figcaption></figure>
by *pS4:nGFP* and *FGR7.0* suggest that both fertilization products suffer from mixed identities in a significant number of *tgs1* ovules. Furthermore, DIC observations and fluorescence analysis for two embryo cell fate reporters, *pS4:nGFP* and *pDRN:GFP*,

indicated that loss of apical-basal polarity (Fig. 5a–e) and aber-
rant cell division patterns in both proper embryos (Fig. 5g–n)
and suspensors (Fig. 5f,o,p) were significantly more common at
2–5 DAP in *tgs1* embryos (27/380 and 29/468 in *tgs1-4* and *tgs1-1*

<figure><img src="image_10.png" /><figcaption>**Fig. 5** Embryogenesis in wild-type (WT) and *tgs1* plants of *Arabidopsis thaliana*. (a–p) Alterations in cell identity and division patterns include loss of apical-basal polarity and fate specification resulting in the formation of extra embryo in the suspensor (b), filamentous embryonic structures (c, d), disorganized embryo (e) and disordered divisions affecting lower suspensor cells (f) and hypophysis cell (o, p). (d) shows *pS4:nGFP* expression. (g–n) Normal (g, j, n) and disordered divisions (arrowheads) in the apical cell (h, i) and daughter cells (k–m), associated occasionally with mislocalization of the *pDRN:GFP* cell identity marker (m, n). (i) shows *pS4:nGFP* expression. (q–u) Formation of extra zygotes (r; *pS4:nGFP* expression) and extra embryos (t, u). Stars indicate supernumerary embryos. Inset in (q) shows *pS4:nGFP* expression. Bars, 5 µm.</figcaption></figure>
5 seeds, respectively) than in WT embryos observed at the same stage (3/233; two-tailed *P* value for Fisher's exact test < 0.0005). More strikingly, while WT fertilized ovules always contained a single embryo (*n* > 500), we observed the formation of two zygotes (4/100) and multiple embryos (14/409) during early (Fig. 5q–u) and late (Fig. S8a) *tgs1* seed development. When sown on germination medium, and consistently with our observations indicating altered embryo patterning, only 67% of mutant (*tgs1-4* and *tgs1-5*) seeds germinated (*n* = 400; WT germination > 95%, *n* > 200; Fig. S8). Out of these, 20% and 29% exhibited defects in primary root and leaf, respectively (developmental defects in WT < 2%; *n* > 200; Fig. S8). Finally, our observations in *tgs1* germinated seeds indicated the generation of suspensor-derived twin embryos (3.1%; Fig. S8b,g). However, we found no evidence for multiple dizygotic embryos, suggesting that only one of the embryos detected during early and late seed development is able to complete development.

## *tgs1* seeds contain triploid embryos

As our observations suggested that mature, unreduced FG occur at low frequency, we asked whether these FG are functional, that is, do *tgs1* seeds contain unreduced embryos? We thus determined DNA contents in nuclei from mature seeds by flow cytometry. In *A. thaliana*, nuclei typically distribute among peaks generated by embryo cells (2× and 4×) and cells of the remanent endosperm (3× and 6×) while maternal tissues from the seed

coat contain no intact nuclei (Matzk *et al.*, 2000). As *pWOX2: CENH3-GFP* sperm nuclei at anthesis showed a five-dot pattern corresponding to the reduced number of chromosomes (Fig. S6h,i), seeds successfully formed from an unreduced, female FG therefore are expected to contain a 3× embryo. First, we characterized nuclei distribution in 17 bulks of four WT seeds, which showed peaks corresponding to 2×, 3×, and > 3× DNA content values (61%, 8% and 31%, respectively; Fig. 6a,d). This indicated that all WT seeds contained a diploid embryo and a small proportion of 3× cells likely from remanent endosperm. The same procedure applied for screening 300 *tgs1-4* seeds and 220 *tgs1-5* seeds (respectively, 75 and 55 bulks) allowed the detection of five bulks showing a significant difference in nuclei proportions compared with estimated mean DNA content values in bulks of WT seeds. Out of these, an increased proportion in 3× cells explained this difference, while the remaining bulk contained a higher proportion of cells with > 3× DNA content value (Fig. 6b–d). Our procedure allowed the detection of 3× embryos at low frequency in mature *tgs1* seeds (i.e. < 1%). However, considering the defects reported during seed development, it is likely that the frequency of functional, unreduced female gametes is higher than that estimated in mature seeds.

## Discussion

Previous experiments in *L. angustifolius* (Taylor *et al.*, 2021) and *P. notatum* (Siena *et al.*, 2014; Colono *et al.*, 2019) supported a

<figure><img src="image_11.png" /></figure>

<table><caption>**Fig. 6** DNA content analyses in WT and *tgs1* nuclei of *Arabidopsis thaliana* seeds. Distribution of signals for nuclei extracted from one bulk of four WT seeds (a), one bulk of four *tgs1-5* seeds with no evidence of 3× embryo (b), and one bulk of four *tgs1-5* seeds likely containing a 3× embryo (c). (d) Counts obtained for three classes of nuclei (2×, 3×, > 3×) from the five *tgs1* seed bulks showing significantly biased nuclei distributions compared with nuclei proportions in WT seeds (*f*<sub>WT</sub>) estimated from 17 bulks (two-tailed *P* value for χ² (2, *n* = 950–7115) < 0.0001). R<sub>2x/3x</sub> shows χ² (1, *n* = 786–4950) values for testing 2×/3× nuclei ratios. ns, ** and *** denote two-tailed *P* values > 0.05, < 0.01, and < 0.001, respectively.</caption><thead><tr><th>Nuclei classes</th><th>f<sub>WT</sub></th><th>tgs1-4/tgs1-4<sub>1</sub></th><th>tgs1-4/tgs1-4<sub>2</sub></th><th>tgs1-4/tgs1-4<sub>3</sub></th><th>tgs1-4/tgs1-4<sub>4</sub></th><th>tgs1-5/tgs1-5</th></tr></thead><tbody><tr><td>2x</td><td>0.616</td><td>1767</td><td>669</td><td>3265</td><td>831</td><td>3570</td></tr><tr><td>3x</td><td>0.082</td><td>691</td><td>117</td><td>347</td><td>119</td><td>1380</td></tr><tr><td>&gt; 3x</td><td>0.302</td><td>458</td><td>167</td><td>650</td><td>667</td><td>2162</td></tr><tr><td>R<sub>2x/3x</sub></td><td>0.883</td><td>634.9***</td><td>7.5**</td><td>16.0***</td><td>0.6<sup>ns</sup></td><td>1242.3***</td></tr></tbody></table>
pathway (Mendes et al., 2020). How TGS1 mediates reproduc-
tive fate determination in the nucellus remains to be elucidated,
but it is attractive to speculate whether TGS1 and STK belong to
the same network acting during early steps of ovule primordium
development, an assumption well supported by the regulation of
STK by the HUA-PEP module, a complex of RNA-binding pro-
teins responsible for pre-mRNA processing (Rodríguez-Cazorla
et al., 2018) and that could partner with TGS1 considering its
activity in transcriptional regulation (Zhu et al., 2001).

Abnormalities in FM specification at the end of meiosis appear
somewhat different from that observed in *Arabidopsis* mutants
affected in this process. In plants overexpressing ARABINO-
GLUCAN PROTEIN18 (AGP18) and in the septuple *interactor/
inhibitor of cyclin-dependent kinase (ick)* mutant, one to four
megaspores at any position can survive, acquire FM fate and,
eventually, produce a FG in *ick* mutants (Demesa-Arévalo &
Vielle-Calzada, 2013; Cao *et al.*, 2018). In contrast, *tgs1* ovules
typically contained tetrads with the two chalazal megaspores spe-
cified for gametophytic development (i.e. positive for pWOX2:
CENH3:GFP; Fig. 2p) while the micropylar spores remained
nonfunctional and degenerated. As shown by ICK inactivation,
the micropylar cell of the meiotic dyad often undergoes an
incomplete second meiotic division in Arabidopsis (Cao
et al., 2018), therefore suggesting that TGS1 repressing activity
requires the completion of the second meiotic division.

Finally, TGS1 expression throughout coenocytic FG develop-
ment was weak until we observed strong GFP signals in the egg cell
(and to some extent in the central cell) after FG cellularization.
This event marks cell fate acquisition in the *Arabidopsis* FG (Susaki
et al., 2021) and, likewise, our data suggest that adopting a strong,
specific TGS1 expression level could also be a marker for egg cell
fate. In support, nuclei misplacement and overproliferation, and
dual cellular identities in micropylar end of *tgs1* FGs substantiate a
role in coordinating gametophytic development and specifying cell
fates in a manner reminiscent of AMP1, ATO, and RKD tran-
scription factors, and of BEL1-LIKE HOMEODOMAIN1,
whose depletion and ectopic expression in the *Arabidopsis* FG,
respectively, result in egg cell fate in synergid cells (Pagnussat
et al., 2007; Moll et al., 2008; Kong et al., 2015; Tedeschi
et al., 2017). Gametic mis-specification did not prevent pollen gui-
dance nor double fertilization, but seeds derived from *tgs1* FGs
showed reduced viability (Table 1) and a variety of defects, includ-
ing mixed cellular identities in fertilization products, abnormal
embryo patterning, reduced proliferation in the endosperm, multi-
ple zygote and embryo development. As paternal *TGS1* alleles do
not rescue defects in *tgs1/+* seeds (Table 1), these abnormal pheno-
types likely resulted from adverse effects of mis-specification in the
FG on the regulation of mechanisms mediating maternal gameto-
phytic control on early seed development (i.e. transcript carryover,
epigenetic reprogramming, cell cycle regulation, and hormonal sig-
naling; Ge et al., 2010; Dresselhaus & Jürgens, 2021; Verma
et al., 2022).

These findings, the versatile functions of TGS1 proteins in
RNA biology and transcriptional regulation and, possibly, other
unknown activities as suggested by the presence of the protein-
protein interaction WW domain, posit RNA methyltransferases as

attractive integrators of the epigenetic, hormonal, and cell cycle
components controlling the female reproductive lineage in plants.
The incomplete penetrance of the defects caused by the two *tgs1*
mutations reported here likely reflects functional redundancy
involving paralogous TGS1-LIKE genes or epistatic interactions
involving unrelated, yet unknown genes. In regard to TGS1 regu-
latory function through splicing of genes required for sporogenesis
in yeast (Qiu *et al.*, 2011), the critical role of alternative splicing in
post-transcriptional regulation of genes essential to plant reproduc-
tion (Cucinotta *et al.*, 2021; H. Li *et al.*, 2021; Kashkan
*et al.*, 2022) might require TGS1, a hypothesis we are currently
examining through RNAseq in reproductive tissues of *A. thaliana*.

In conclusion, our work indicates that TGS1 contributes to the evolutionary acquisition of monospory (i.e. the formation of a single FG from the most chalazal spore), a dominant trait among angiosperms likely canalized toward the prevention of multiple female germline development (Webb & Gunning, 1990; Bachelier & Friedman, 2011). TGS1 depletion in *Arabidopsis* recapitulates, to varying degrees, the reproductive phenotypes unleashing the canalizing mechanisms that operate during female germline development and, in consequence, promotes favorable conditions for the emergence of an aposporous developmental pathway in a sexually reproducing species. Heterochrony plays an essential role in living organism diversification and has been essential for gametophyte development evolution (Buendía-Monreal & Gillmor, 2018). Here, we postulate that apospory may result from a heterochronic shift allowing MMC specification and gametophytic fate, two cellular decisions separated in time during female germline progression, to occur concurrently. Therefore, TGS1 appears to be a candidate for the genetic control of critical fate decisions that govern female germline development in plants. Our findings open the wedge for investigating how the interplay between TGS1 and key pathways for plant reproduction involved in small RNAs, phytohormones regulation, and cell-cell communication (Lora et al., 2019; Pinto et al., 2019; Petrella et al., 2021) regulates cell fate decision during plant female reproduction.

Acknowledgements

We thank Dr Rita Groß-Hardt, Dr Gerd Jürgens, the NASC/
ABRC *Arabidopsis* stock centers for mutants and marker lines,
and Daphné Autran for helpful comments on the manuscript.
We also acknowledge the imaging facility MRI, member of the
national infrastructure supported by the French National
Research Agency (ANR-10-INBS-04, 'Investments for the
future'). BS was supported by Agropolis Fondation under the
reference ID 1502-008 through the Investissements d'avenir pro-
gram (Labex Agro: ANR-10-LABX-0001-01) and the European
Union's H2020 research and innovation program under the
Marie Skłodowska-Curie grant agreement no. 645674, Project
PROCROP. LAS and OL were supported by the European
Union's research and innovation program under the Marie
Skłodowska-Curie grant agreement no. 645674 (Project PRO-
CROP) and no. 872417 (Project MAD). JPAO, LAS, and SCP
were supported by the National Agency for Scientific and
Technological Promotion (ANPCyT), Argentina (PICT-2017-1956 and PICT 2019-03414). JPAO, LAS, and SCP are research staff members of CONICET, Argentina, and JMV was supported by a PhD grant from CONICET.

Competing interests

None declared.

Author contributions

OL and LAS conceived and designed the study. BS, CM, JMV, LAS, MI and OL conducted the experiments. BS, CM, JMV, JPAO, LAS, MI, OL and SCP contributed to analyze the data. LAS, MI and OL prepared the figures. LAS and OL wrote the manuscript and BS, CM, JMV, JPAO, MI and SCP contributed to its revision and editing.

ORCID

Mathieu Ingouff https://orcid.org/0000-0002-6106-4564

Olivier Leblanc https://orcid.org/0000-0003-3641-1875

Caroline Michaud https://orcid.org/0000-0002-0620-2442

Juan Pablo A. Ortiz https://orcid.org/0000-0001-8460-6154

Silvina C. Pessino https://orcid.org/0000-0002-0200-8706

Benjamin Selles https://orcid.org/0000-0002-9260-9344

Lorena A. Siena https://orcid.org/0000-0003-3738-9075

Data availability

The data that support the findings of this study are available in the main text and figures and from Supporting Information.

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Supporting Information

Additional Supporting Information may be found online in the Supporting Information section at the end of the article.

**Fig. S1** Characterization of *Arabidopsis thaliana* T-DNA insertion lines for *AtIg45231*.

**Fig. S2** Multiple sequence alignment of plant proteins homologous to yeast TGS1.

**Fig. S3** Detailed phylogenetic tree of Fig. 1(c).

**Fig. S4** GUS patterns in reproductive tissues of *Arabidopsis thaliana* plants carrying *pAtTGS1:AtTGS1-GUS*.

**Fig. S5** Formation of binucleated female gametophyte in *tgs1* ovules at meiosis in *Arabidopsis thaliana*.

**Fig. S6** *pWOX2-CENH3-GFP* expression patterns during reproductive development in *Arabidopsis thaliana*.

**Fig. S7** Identity defects in *tgs1* gametophytes in *Arabidopsis thaliana*.

**Fig. S8** Phenotypes of *tgs1* embryos and germinated *tgs1* seedlings in *Arabidopsis thaliana*.

**Notes S1** Multiple alignment of plant TGS1 proteins. This file opens in a text editor or using the MView utility.

**Notes S2** Amino acid sequences of conversed blocks used for phylogenic tree inferences. This file opens in a text editor or using the MView utility.

**Table S1** Sequences orthologous to yeast TGS1 by species.

**Table S2** List of primers.

Please note: Wiley is not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the *New Phytologist* Central Office.