# The CELL NUMBER REGULATOR SIFWL5 protein regulates aerial vegetative growth in tomato, by promoting cell expansion

Arthur Beauchet, Lucie Ehrhard, Lina Boutaleb, Valérie Rofidal, Nathalie Gonzalez, Christian Chevalier, Norbert Bollier

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Arthur Beauchet, Lucie Ehrhard, Lina Boutaleb, Valérie Rofidal, Nathalie Gonzalez, et al.. The CELL NUMBER REGULATOR SIFWL5 protein regulates aerial vegetative growth in tomato, by promoting cell expansion. Journal of Experimental Botany, 2025, 10.1093/jxb/eraf444. hal-05325832

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Distributed under a Creative Commons Attribution 4.0 International License
Title: The CELL NUMBER REGULATOR SIFWL5 protein regulates aerial vegetative growth
in tomato, by promoting cell expansion

Arthur Beauchet<sup>†,‡,1</sup>, Lucie Ehrhard<sup>‡,1</sup>, Lina Boutaleb<sup>1</sup>, Valérie Rofidal<sup>2</sup>, Nathalie Gonzalez<sup>1</sup>, Christian Chevalier<sup>*†</sup> and Norbert Bollier<sup>1</sup>

<sup>1</sup>INRAE, Université Bordeaux, UMR1332 Biologie du Fruit et Pathologie, Villenave d'Ornon F-33140, France

<sup>2</sup>IPSiM, CNRS, INRAE, Institut Sup Agro, Université Montpellier, Montpellier F-34060, France

<sup>†</sup>Present address: VIB-UGent for Plant Systems Biology, Ghent University, 9052 Ghent, Belgium
<sup>‡</sup>The authors contributed equally to this work

* *To whom correspondence should be addressed.* E-mail: christian.chevalier@inrae.fr

Authors' E-mail: arbea@psb.vib-ugent.be; lucie.ehrhard@inrae.fr; lina.boutaleb@inrae.fr; valerie.rofidal@inrae.fr; nathalie.gonzalez@inrae.fr; christian.chevalier@inrae.fr and norbert.bollier@inrae.fr

Date of submission = 09/03/2025

Number of figures = 7

Number of tables = 1

Number of words = 5724 (Introduction + Results + Discussion + Acknowledgements)

Number of supplementary data = 8 figures, 4 tables

Running title: SIFWL5 regulates aerial vegetative growth via cell expansion

**Highlight:** The FW2.2-LIKE/CELL NUMBER REGULATOR protein SIFWL5 is localized at plasmodesmata and regulates aerial vegetative growth in tomato, via cell expansion.
**1 Abstract:**

The *FW2.2-LIKE/CELL NUMBER REGULATOR (FWL/CNR)* gene family comprises PLAC8 domain-containing membrane-associated proteins and was named in reference to its founding member, the *FW2.2* gene, which determines fruit size in tomato via a negative regulation on cell divisions. The function of PLAC8 domain-containing FWL/CNR proteins in plants remains largely unexplored. Only recently has the molecular and cellular mechanism of FW2.2 been described as regulating plasmodesmata-mediated cell-to-cell communication. In the present study, we provided a functional analysis of *SIFWL* genes in tomato, aiming at investigating any direct role in the control of organ growth. Based on a combination of molecular and cellular approaches, we selected three SIFWL proteins, namely SIFWL2, -4 and -5, which are localized at the plasma membrane. Gain- and loss-of-function transgenic plants were generated to explore their putative role as regulators of organ growth in tomato. This allowed us to shed light more specifically on the critical involvement of SIFWL5 in leaf and hypocotyl development. We show here that SIFWL5 is localized at plasmodesmata, and that it regulates leaf size and morphology, and hypocotyl growth, by promoting cell expansion, as unexpected for a CELL NUMBER REGULATOR protein. This original finding underscores further the importance of some FWL/CNR family members in growth regulation.

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**19 Keywords:** cell expansion, CELL NUMBER REGULATOR, FW2.2-LIKE, leaf growth, hypocotyl growth, plasma membrane, plasmodesmata, tomato

**21 Introduction**

The *FW2.2-LIKE/CELL NUMBER REGULATOR (FWL/CNR)* gene family (Guo *et al.*, 2010; Thibivilliers *et al.*, 2020) was named in reference to its founding member, the *FW2.2* gene (standing for Fruit Weight QTL on chromosome 2, number 2), which underlies the major quantitative trait locus (QTL) governing fruit size in tomato (Alpert *et al.*, 1995; Frary *et al.*, 2000). The *FWL/CNR* gene family encompasses hundreds of related sequences in the plant, animal, and fungal reigns (Guo *et al.*, 2010), with a large diversity in protein size ranging from a hundred to several hundred amino acids. The FWL/CNR proteins are usually described as membrane-associated Pro- and Cys rich proteins, which harbor as a principal feature, the presence of a conserved structural domain named PLAC8 (Placenta specific 8), originally identified in a protein specifically expressed in the trophoblast giant cells and spongiotrophoblast layer of the placenta from mouse (Galaviz-Hernandez *et al.*, 2003). The PLAC8 domain is composed of one or two hydrophobic segments, predicted to form two putative transmembrane

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(TM) α-helices, and characterized by the presence of Cys-rich motifs of the CLXXXXCPC or
CCXXXXCPC type (Song et al., 2004; Beauchet et al., 2021).

Three main functions have been assigned to PLAC8 domain-containing FWL/CNR proteins in
plants: the regulation of organ size, especially in fruits, cadmium resistance, and metal ion
homeostasis (Beauchet et al., 2021). Indeed, the original FW2.2 protein regulates negatively cell
divisions in the pre-anthesis ovary and young developing fruit, thereby accounting for its
quantitative effects on final fruit size via the modulation of cell number (Nesbitt and Tanksley,
2001; Cong et al., 2002; Liu et al., 2003). Functional analyses of FWL/CNR proteins suggest
that this role in the regulation of organ growth, primarily through their effects on cell division, is a
highly conserved feature in both monocotyledon and dicotyledon plants (Dahan et al., 2010; Guo
et al., 2010; Li and He, 2015; Qiao et al., 2017; Ruan et al., 2020). Interestingly and related to
this function, the PLAC8 protein has been determined as well to be involved in organ
development and cell division control during tumorigenesis in mammals (Cabreira-Cagliari et al.,
2018; Mao et al., 2021). However, the precise molecular mechanism underlying the role of
FWL/CNR proteins in regulating organ growth linked to cell division/cell cycle control remain
largely unexplored.

Recently, Beauchet et al. (2024) made a major breakthrough towards the identification of the
molecular and cellular mode of action of FW2.2 in tomato. In this study, FW2.2 was
demonstrated to be a membrane-anchored protein that locates at plasmodesmata (PD) and
regulates cell-to-cell communication by modulating PD transport capacity during fruit
development, via the regulation of callose deposition at PD (Beauchet et al., 2024). Whether this
function is conserved in FWL/CNR proteins from other plant species remains to be determined.
Nevertheless, several clues seem to comfort this functionality. Indeed, the closest ortholog of
FW2.2 in Arabidopsis, namely AtPCR2, is highly enriched at PD (Brault et al., 2019). The
soybean GmFWL1 and GmFWL3 proteins are localized to membrane microdomains, and
interacts with structural proteins of the membrane microdomains, but also with proteins involved
in the metabolic process of callose deposition, such as callose synthases (Qiao et al., 2017;
Cervantes-Pérez et al., 2024), alike FW2.2 in tomato fruit (Beauchet et al., 2024).

An updated phylogenetic analysis performed on 13 different plant genomes released in
recent years allowed the identification of 134 members in the FWL/CNR family, with a total
number of twelve FWL genes including FW2.2 in tomato (Thibivilliers et al., 2020). This list has
been recently extended up to 21 members (Ran et al., 2023), providing a valuable resource for
the characterization of FWLs in tomato.
In the present study, we provided the first functional analysis of SIFWL genes in tomato. We aimed at investigating whether SIFWLs can play a direct role in the control of organ growth. First, we re-investigated the expression profiles of SIFWLs in different organs of tomato plants and during fruit development, and analyzed their subcellular localization. Based on this combination of molecular and cellular approaches, we selected three SIFWL genes, namely SIFWL2, -4 and -5, and generated gain- and loss-of-function transgenic plants to explore their putative role as regulators of organ growth and development in tomato. This allowed us to shed light more specifically on the critical involvement of SIFWL5 in the regulation of cell expansion during leaf development and hypocotyl growth, further underscoring the importance of some FWL/CNR family members in growth regulation.

## Materials and methods

### Plant materials and growth conditions

Tomato (Solanum lycopersicum cv. AC) and tobacco (Nicotiana benthamiana) plants were grown in soil in a greenhouse under the following conditions: 16 h day/8 h night cycle, using a set of 100 W warm white LED projectors providing an irradiance of 100 µmol m⁻² s⁻¹ at the level of canopy. The light spectrum was constituted by equivalent levels of blue irradiation (range 430–450 nm) and red irradiation (640–660 nm). For in vitro culture, tomato seeds were sterilized for 10 min under agitation in a solution of 3.2% bleach. Seeds were then washed three times with sterile water and dried under a laminar flow hood. Seeds were sowed in Murashige and Skoog medium (1/4 MS) and transferred in a growth chamber under the following conditions: 16 h day/8 h night cycle, 22°C/20°C day/night, using white light (Osram L36 W/77 Fluora 1400 lm) providing 80 to 100 µE m⁻² s⁻¹ intensity light at the stirring plate.

### Phylogenetic analysis and tools for the prediction of the CNR/FWL structure and topology

The 21 SIFWL protein sequences from tomato were mined from Thibivilliers et al. (2020) and Ran et al. (2023), and aligned using MEGA11 Muscle alignment (Tamura et al., 2021). Values for 1000 bootstrap replicates are shown on each branch.

DeepTMHMM (https://dtu.biolib.com/DeepTMHMM) (Hallgren et al., 2022) was used to predict the presence of transmembrane helix in SIFWL proteins. The 3-dimensional structure of the full-length SIFWL2, -3 and -5 proteins was obtained using Colabfold (Mirdita et al., 2022) The PPM 3.0 Web Server (https://opm.phar.umich.edu/ppm_server3_cgopm) (Lomize et al., 2022)
1 was used with default parameters and plasma membrane (plants) type to predict the topology
2 and insertion of SIFWL2, -3 and -5 in the plasma membrane.

3

4 Vector constructs and plant transformation

5 Vectors for the overexpression of SIFWLs in plants were generated using the Gateway® cloning
6 system (Invitrogen, Carlsbad, CA, USA), following manufacturer's instruction. The FWLs full-
7 length coding sequence was amplified from cDNAs prepared from various tomato tissues (cv.
8 AC) using PrimeSTAR MAX DNA polymerase (TAKARA BIO Inc., Kusatsu, Japan) and primers
9 including the attB sites (Supplemental Table S1). The resulting PCR products were cloned into
10 the corresponding Gateway vectors described in Supplemental Table S2. For CRISPR/Cas9
11 mutagenesis, constructs were assembled using the Golden Gate cloning method according to
12 previous method (Beauchet et al., 2024). Transgenic plants were generated by Agrobacterium
tumefaciens (strain C58C1) mediated transformation using explants of tomato cotyledons as
13 described (Swinnen et al., 2022).
14

15

16 RNA extraction and RT-qPCR analysis

17 Total RNA was isolated from cotyledons, hypocotyls, shoot apical meristems, leaves, roots,
18 flowers and pericarp tissues from fruits harvested at different developmental stages (5, 10, and
19 15 DPA), using TRIzol reagent (Invitrogen) in combination with RNeasy Plant Mini Kit (Qiagen)
20 following the manufacturers' instructions. RNase-free DNase (Qiagen) treatment was performed
21 on each sample. Reverse transcription was performed using the iScript™ cDNA Synthesis Kit
22 (Bio-Rad, Hercules, CA). Real-time PCR was performed using Gotaq® qPCR mastermix
23 (Promega, Madison, WI) and a CFX 96 real-time system (Bio-Rad). qPCR primers were
24 designed with PerlPrimer software (Marshall, 2004) to overlap 2 exons in order to limit genomic
25 DNA amplification (Supplemental Table S2) and amplify a 80 to 200 bp-long amplicon, with a
26 Tm of 60°C. The transcript levels of the expressed genes were normalized to that of the
27 housekeeping genes: SITUBULIN (Solyc04g081490) in combination with SINUDK
28 (Solyc01g089970) for fruit samples, or with SIEIF4α (Solyc12g095990) for other tissue samples.
29

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30 Subcellular localization analysis of SIFWL proteins

31 The subcellular localization of SIFWLs fused to YFP was observed using confocal imaging
32 performed on a Zeiss LSM 880 confocal laser scanning microscope equipped with fast AiryScan,
33 using a Zeiss Plan apochromat 10x (NA 0.45) or a C PL APO x63 (NA 1.4) oil-immersion
34 objective.
The top three leaves of three independent *N. benthamiana* plants (6–8 weeks old) were infiltrated with 35S::SIFWLs-YFP constructs and plasmolyzed using 0.4 M sorbitol for 15 min before observation. Co-infiltrations with SP-mTagBFP2-HDEL as a marker of ER and mScarlet-I as a marker of both cytosol and nucleus localization were performed to ascertain the localization pattern. Staining with FM4.64 at a final concentration of 4 µM was used as a control for PM localization (Bolte *et al.*, 2004). For FM4.64 imaging, excitation was performed at 561 nm and fluorescence emission was collected at 630-690 nm. For YFP imaging, excitation was performed at 514 nm and fluorescence emission collected at 520-580 nm. For mTagBFP2 imaging, excitation was performed at 405 nm and fluorescence emission was collected at 440-490 nm. For mScarlet-I imaging, excitation was performed at 561 nm and fluorescence emission was collected at 570-640 nm.

The localization of SIFWL2, -4 and 5 at PM and PD was performed in leaves of tomato 35S::SIFWL2-YFP, 35S::SIFWL4-YFP and 35S::SIFWL5-YFP transgenic plants. Similarly, leaf epidermal cells were plasmolyzed using 0.4 M sorbitol prior to observation. Staining with aniline blue (Biosupplies, Victoria, Australia) was performed by infiltration of a 0.0125% solution; excitation was performed at 405 nm and fluorescence emission collected at 420-480 nm. The calculation of PD index was determined by calculating the fluorescence intensity of SIFWL2-YFP, SIFWL4-YFP and SIFWL5-YFP at plasmodesmata and at PM as described previously (Grison *et al.*, 2019).

## Phenotypic characterization

Plants were cultivated randomly side-by-side with WT plants. Flowers were vibrated every day to ensure optimal self-pollination. Seven flowers per inflorescence were maintained to ensure proper development of fruit per inflorescence. Fruits from four to six plants of each genotype of two biological replicates were used to determine fruit weight at the breaker stage of fruit development. Fruits were weighted and measured using a caliper. The number of measurements ranged from n=50 to n=200 depending on the number of fruits produced by the different transgenic plants. For root and hypocotyl length measurements, seedlings were grown on MS1/4 in 245 x 245 x 20 mm Nunc tissue culture plates placed vertically. Pictures of seedlings were digitized using a scanner every day and root and hypocotyl length were measured using ImageJ software. For leaf surface phenotyping, pictures of full-grown leaves were taken using a Nikon D5300 and analyzed by intensity threshold filtering in ImageJ. For the analysis of leaf epidermal cell size, leaflets were cleared with 70% ethanol for several days before staining with Calcofluor white M2R stain 0.1 % (w/v) for 48 h. Leaflets fragments were
observed with a Zeiss Axioimager epifluorescence microscope equipped with a 20x (NA 0.5) using a DAPI-BP filter and a Cool Snap HQ2 CCD Camera. For the measurements of hypocotyl epidermal cell size, freshly harvested hypocotyls were fixed in 4% (w/v) paraformaldehyde (PFA) in 1X PBS under vacuum for 30 min, rinsed 3 times with 1X PBS and incubated in Clearsee solution for at least 2-weeks. Samples were then stained with 0.1 % (w/v) Calcofluor white M2R for 48 h in fresh Clearsee. Central epidermal cells of each hypocotyls were imaged with a Zeiss LSM 880 confocal microscope using a Plan apochromat 10x dry objective (NA 0.45); excitation was performed at 405 nm (0.2% laser power) and fluorescence emission was collected between 430 to 490 nm.

## Immuno-labelling of callose

The level of callose deposition was quantified in leaves from four week-old plants, by immunofluorescence labeling using a callose-specific antibody, as described in Beauchet *et al.* (2024). The signal intensity was subsequently quantified as a proxy of callose deposition at PD, by integrating the gray value of all the pixels above the same threshold. A minimum of six measurements was performed at least on 5 sections from at least three different leaves from different plants, and the experiment was repeated twice.

## Co-immunoprecipitation and mass-spectrometry analysis

Total protein extracts from 100 mg of 35S::SIFWL5-YFP leaf tissue were prepared using the following buffer: 1X PBS, cOmplete Protease Inhibitor Cocktail tablets (Roche, Mannheim, Germany) and 1% Triton X-100. Samples were incubated in the extraction buffer at 4°C for 30 min with agitation, and then centrifuged (16000g, 10 min, 4°C). Prior to co-immunoprecipitation, western blotting was used to check the presence of the expressed tagged-SIFWL5-YFP protein in the supernatant. The supernatant containing the resuspended proteins was then used for immunoprecipitation assay using anti-GFP microbeads provided in the µMACS Epitope Tag Protein Isolation Kit according to the manufacturer's protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). Protein concentration was determined by Bradford assay according to the manufacturer's instructions (Pierce Coomassie Plus (Bradford) Assay kit; Thermo Scientific). Approximately, 500 µg of soluble proteins were loaded for each co-IP assay. The LC-MS-MS experiment was carried out as reported previously (Beauchet *et al.*, 2024).

The GO enrichment analysis was carried out on PLAZA 5.0 (Van Bel *et al.*, 2022) using the Plaza workbench, with a significance threshold of 0.05 and without any data filter.
The mass spectrometry proteomics data have been deposited to the ProteomeXchange
Consortium via the PRIDE (Perez-Riverol et al., 2022) partner repository with the dataset
identifier PXD061438.

## Results

### Phylogenetic classification and gene expression profiles of SIFWLs

A recent genome-wide analysis of the SIFWL family identified a total of 20 FW2.2 homologous sequences in tomato (Ran et al., 2023). These authors named the different genes according to their location on the tomato chromosomes, not taking into account the anteriority of the SIFWL gene classification (although incomplete) proposed by Thibivilliers et al. (2020). To avoid additional confusion and to compel to the principle of anteriority, we proposed to keep the latter gene annotation and to assign ascending gene names to the 9 new identified SIFWL sequences according to their phylogenetic relationship to FW2.2 (Table I).

A phylogenic tree was thus generated using the full-length sequences of FW2.2 and the 20 homologous sequences from tomato (Figure 1A). The SIFWLs fall into two distant clades: the first clade includes FW2.2 and the eleven sequences identified by Thibivilliers et al. (2020), with the addition of SIFWL12; the second clade encompasses SIFWL13 to SIFWL20. Remarkably, SIFWLs from the first clade display a highly similar protein homology, with the exception of SIFWL4, SIFWL7, -8 and -12 which harbor a long N-terminal extension upstream of the conserved PLAC8 domain. SIFWLs from the second clade differ from the first one by a longer sequence, a greater distance between the two conserved motifs of the PLAC8 domain and/or the presence of two or more predicted transmembrane (TM) α-helices as revealed by the use of the transmembrane topology prediction tool DeepTMHMM (Hallgren et al., 2022) (Figure 1A). It is noteworthy that SIFWL9 occupies an intermediate position between the two subgroups, with a relatively smaller sequence than that of FW2.2 and the presence of two TM domains.

**Figure 1B** describes the primary structure of the conserved PLAC8 domains in the 20 SIFWL proteins. The CLXXXXCPC motif is only conserved in FW2.2, SIFWL8 and SIFWL15, while SIFWL13 and -14 harbors a CLXXXXAPC sequence. SIFWL2, -3 and -10 harbors the CCXXXXCPC sequence. The CLXXXXCPC or CCXXXXCPC motif diverges more or less in the other SIFWL proteins, especially for the two first amino acids of the sequence. The second motif at the C-terminal part, the so-called 'QXXRELK' motif, appears much more conserved among the 20 SIFWL proteins.

To provide an overview of the expression pattern in tomato, the levels of transcript accumulation for SIFWL genes were monitored by RT-qPCR in various organs and fruit
developmental stages from the tomato cultivar 'Ailsa Craig' ('AC') (Supplemental Figure S1).

This analysis showed that a large diversity of expression profiles was observed among the tested SIFWL genes, not only in terms of organs, but also in terms of level of transcript accumulation. For instance, SIFWL3, SIFWL14, and SIFWL15 exhibited much higher expression levels, ~a hundred-fold more, than all the other SIFWL genes. Most of the SIFWL genes are expressed to various levels throughout fruit development. SIFWL3, SIFWL9, SIFWL10 and SIFWL15 were predominantly expressed in fruits, with SIFWL3 and SIFWL15 having the highest expression. FW2.2, SIFWL1 and SIFWL4 were highly expressed in roots and to a lesser extent in fruit. SIFWL2 was predominantly expressed in cotyledons, and to a lesser extent in hypocotyls, leaves and flowers, and slightly in roots. SIFWL5 showed high expression in both roots, cotyledons and to a lower extent in leaves and hypocotyls. SIFWL7 and SIFWL8 were more specifically expressed in flowers. SIFWL13, SIFWL14, SIFWL16, SIFWL17 and SIFWL18, were broadly expressed across nearly all tissues.

## Sub-cellular localization of SIFWL proteins

The subcellular localization of SIFWL proteins was first investigated in *Nicotiana benthamiana* leaves, using transient expression assays. Constructs aimed at expressing the SIFWL proteins fused to YFP at the C-terminal end, under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter were thus generated (referred to as 35S::SIFWLX-YFP). This type of constructs was chosen based on our previous work, showing that the sub-cellular localization of FW2.2 was independent from the position of YFP at the C-terminal or N-terminal end of the protein (Beauchet *et al.*, 2024). Figure 2 displays the data obtained for eleven out of twenty SIFWLs. The following SIFWLs were not included in this analysis for the following reasons: (i) we could not state in the subcellular localization of SIFWL3, due to a lack of reproducibility in the results; (ii) we were unable to generate the constructs fusing the coding sequence for SIFWL12, -13, -14, -16, -17 to YFP for cloning technical reasons; (iii) SIFWL18, -19 and -20 were excluded from the study, because of their too distant phylogenetic relationship with the other SIFWLs.

For the tested SIFWLs, three distinct patterns of subcellular localization were observed. First, SIFWL1, -6, -9, -10, -11 and -15 were distributed within the cell in a reticular pattern, consistent with a localization at the endoplasmic reticulum (ER). This ER localization was confirmed through co-infiltration with the ER marker SP-mTagBFP2-HDEL (J. Dragdwidge, pers. communication) and subsequent co-localization analysis (Figure 2A).
Second, SIFWL7 and -8 were found to localize to both the cytosol and the nucleus. The use of co-infiltrated mScarlet-1 marker, a monomeric constitutive red fluorescent protein (Bindel et al., 2017), confirmed this localization (Figure 2B).

Third, SIFWL2, SIFWL4 and SIFWL5 were targeted to the plasma membrane (PM), as witnessed by staining with FM4.64, a membrane-specific dye (Bolte et al., 2004), following plasmolysis induced by a 0.4 M sorbitol treatment (Figure 2C).

We next investigated the topology of these three SIFWLs at the PM using currently available prediction tools. First, the 3D structure of these PM-localized SIFWLs was predicted using ColabFold software (Mirdita et al., 2021) based on AlphaFold 2.0 (Jumper et al., 2021) and MMseqs2 (Steinegger & Soding, 2017). Second, the insertion in the plasma membrane was modelized using the PPM 3.0 Web Server (Lomize et al., 2022) (Supplementary Fig. S2). These predictions revealed that the 3D structure of SIFWL2, SIFWL4 and SIFWL5 present similarities to that from FW2.2 (Beauchet et al., 2024), such as the absence of any transmembrane domain and their N- and C-terminal parts predicted to be folded on the same side of the protein. These predictions suggest that they do not cross the PM, but are most probably anchored in the PM.

## In planta subcellular localization of SIFWL2, SIFWL4 and SIFWL5

Owing to their localization at the PM, we performed an *in planta* analysis of the subcellular localization of SIFWL2, SIFWL4 and SIFWL5, aimed at investigating whether these three SIFWLs are also enriched at PD in tomato, alike FW2.2 (Beauchet et al., 2024). For this purpose, we generated stable transgenic lines expressing SIFWL2, SIFWL4 and SIFWL5 fused to YFP at the C-terminal end, under the control of the 35S promoter in the tomato cultivar 'AC' (lines referred to as 35S::SIFWL2-YFP, 35S::SIFWL4-YFP and 35S::SIFWL5-YFP plants).

The localization of SIFWL2-YFP, SIFWL4-YFP and SIFWL5-YFP at the PM of tomato leaf cells was confirmed (Figure 3A). In addition, SIFWL5-YFP was found to localize at the PM according to a pattern of punctate spots at the cell periphery, suggesting that SIFWL5-YFP was enriched at nanodomains. Staining with aniline blue (AB) to reveal callose deposition as a marker of PD, revealed that only SIFWL5-YFP co-localized with AB, as shown by the overlapping signal intensity plots (Figure 3A), thus indicating a localization at PD. Next, we determined the plasmodesmata enrichment ratio, named 'PD index', which corresponds to the SIFWLs-YFP fluorescence intensity at PD vs that at the cell periphery, to quantify the enrichment of SIFWLs-YFP at PD, as previously described (Beauchet et al., 2024) (Figure 3B). While the PD index for SIFWL2-YFP and SIFWL4-YFP was equal to 1, a high PD index from 1.5 to 1.7
1 was measured in leaf cells of 35S::SIFWL5-YFP plants, thus demonstrating that only SIFWL5
2 out of the three SIFWLs tested was enriched at PD.

3
4 In planta functional analysis of SIFWL2, SIFWL4 and SIFWL5
5 To analyze the function of SIFWL2, SIFWL4 and SIFWL5 in vegetative and reproductive
6 development, gain- and loss-of-function plants were generated in the tomato cultivar 'AC'.
7 SIFWL2, SIFWL4 and SIFWL5 were overexpressed constitutively and ectopically, using the 35S
8 promoter (gain-of-function plants referred to as 35S::SIFWL2, -4 and -5 respectively). For each
9 gene, three T2 lines were selected with medium- to very high levels of SIFWL2, -4 and -5
10 overexpression in leaves, ranging from 3-fold more to 150-fold more (Supplementary Fig. S3).
11 Alternatively, SIFWL2, SIFWL4 and SIFWL5 were knocked out using the CRISPR/Cas9
12 technology. Two single-guide RNAs (sgRNAs) per gene were designed close to the start codon,
13 in order to create a frameshift or an early stop codon that would result in a dysfunctional protein
14 (Supplementary Fig. S4). For each gene, we selected three independent T0 transgenic lines
15 displaying homozygous mutations, either deletions or insertions, for subsequent phenotyping of
16 vegetative and reproductive development. These loss-of-function plants were referred to as CR-
17 SIfw2, -4 and -5 hereafter.

18 The phenotypic analysis of the different transgenic plants is shown in **Figure 4**. The
19 overexpression of SIFWL2, SIFWL4 and SIFWL5 did not induce any significant effects on both
20 reproductive and vegetative development. Only one line out of three for the 35S::SIFWL2 and
21 35S::SIFWL4 constructs, namely 35S::SIFWL2#2 and 35S::SIFWL4#2, produced smaller fruits (-
22 20% when compared to WT) (Figure 4A). No significant impacts could be detected either on
23 root length (Figure 4B) or leaf surface (Figure 4D), and the sole 35S::SIFWL2#2 line was
24 negatively affected for hypocotyl length, with a reduction in average of 20% when compared to
25 WT (Figure 4C).

26 The most noticeable effects were obtained for CRISPR knockout mutants. Two out of three
27 SIFWL2 mutant lines (namely CR-SIfw2#1 and CR-SIfw2#2), and the sole CR-SIfw5#3 line
28 displayed a statistically significant reduction in fruit weight (respectively -17%, -42% and -24% in
29 average when compared to WT) (Figure 4A). All three CR-SIfw4 knockouts lines were affected
30 for root length with an increase from 15% to 23% in average, when compared to WT plants,
31 while CR-SIfw2 and -5 lines were not affected (Figure 4B and Supplemental Figure S5). In
32 plants harboring non-functional SIFWL5 alleles, the length of the hypocotyl in all three CR-SIfw5
33 lines was reduced from 22% to 38% in average, when compared to WT (Figure 4C). More
34 remarkably, all three CR-SIfw5 lines displayed a marked reduction in leaf surface (from 32% to
47% in average) (Figure 4D), which suggests that the suppression of SIFWL5 alters the overall vegetative development of aerial organs, as root development was not affected.

To the exception of SIFWL2, these results suggested that vegetative growth and development in tomato are regulated by SIFWL4 in roots and more remarkably, by SIFWL5 in leaves and hypocotyls.

## SIFWL5 participates in the control of leaf and hypocotyl growth via cell expansion

We then proceeded to a fine phenotyping of the leaf morphology in all three *CR-SIfw5* lines since the overall leaf surface was reduced compared to the WT. The fully expanded compound leaf of the cultivated tomato cultivar 'AC' is made up of a terminal leaflet (TL) and several lateral leaflets attached to the rachis (Figure 5A). According to the sequential order and position in the rachis, the lateral leaflets can be further categorized into primary (PL), secondary (SL) and intercalary leaflets (IL).

When compared to WT leaves, the leaf morphology of all three *CR-SIfw5* lines was affected, as the production of PL-3, IL-1, IL-3 and SLs was largely reduced (Figure 5B). The *CR-SIfw5#1* line displayed systematically the most altered leaf composition with the lowest number of leaflets. Not only this reduction in the leaflet number was responsible for the overall reduction in leaf surface observed in all three *CR-SIfw5* lines compared to WT (Figure 5C), but also the reduction in the surface of TL, PL and IL contributed to the phenotype (Figure 5D). This reduction in leaflet surface and overall leaf surface in the three *CR-SIfw5* lines was due to a reduction in cell size ranging from 17 to 46% in average (Figure 5E and 5F). The expression levels of *EXPANSIN A* (*EXPA*) genes used as cell expansion markers (Cosgrove, 2000) were further examined in leaves 3 of six-week old plants of *CR-SIfw5* lines by RT-qPCR. Among the different *EXPA* genes expressed in tomato leaves (Lu et al., 2016) (Supplemental Figure S6A), the expression levels of three genes, namely *SIFEXPA7*, *-A10* and *-A12*, were significantly lower in the three mutant lines compared with WT (Figure 5G), thus accounting for the alteration of the cell expansion process when the function of SIFWL5 is suppressed.

Since hypocotyl growth was also affected in the *CR-SIfw5* lines (Figures 4C and 6A), we next investigated whether the phenotype originated from a defect in cell growth. Clearly, the size of epidermal cells in the hypocotyl of 10 days-after-germination (DAG) seedlings was significantly reduced in all *CR-SIfw5* lines compared to WT (Figure 6B), from 21 to 36% in average (Figure 6C). Here too, *CR-SIfw5#1* was the most affected line. Similarly to what was observed in leaves, only three *EXPA* genes among those expressed in stems (Lu et al., 2016) (Supplemental Figure S6B), namely *SIFEXPA3*, *-A7* and *-A18*, were significantly reduced in all
CR-SIFWL5 lines (Figure 6D). The transcript levels of SIFWL5 and SIFWL6 displayed the strongest down-regulation.

## SIFWL5 pull-down reveals plasma membrane- and plasmodesmata-related proteins

The elucidation of SIFWL5 functional role in leaves and at PD relied on a deeper characterization at the biochemical level. For this purpose, we performed an immunoprecipitation followed by tandem-mass spectrometry (IP-MS/MS) on 35S::SIFWL5-YFP leaves to identify interacting protein partners of SIFWL5. The relevance of this proteomics approach required to determine the most appropriate leaf developmental stage to harvest, so that the natural interacting protein partners are present in the protein extracts. Hence, the expression level of SIFWL5 was monitored in the different leaves of 4-week old plants (Supplemental Figure S7). Four-week old plants produced up to 8 leaves: from the newly formed ones, e.g. leaves 8 and 7 emerging from the meristem, to the oldest fully-developed ones, e.g. leaves 2 and 1. The highest expression of SIFWL5 was found in leaves 6, 5 and 4, when leaf growth is highly sustained by cell expansion. Protein extracts were then prepared from these three different stages and pooled prior to immunoprecipitation.

The IP-MS/MS experiment resulted in the identification of 62 proteins that co-immunoprecipitated with SIFWL5, which were significantly enriched in the 35S::SIFWL5-YFP sample when compared to 35S::NLS-GFP-GUS used as a control (Figure 7A, Dataset 1). A Gene Ontology (GO) term enrichment analysis for the 62 identified proteins was performed (Figure 7B), showing that they fall into the three types of GO domains: cellular components (CC), molecular functions (MF) and biological processes (BP). Notably, the CC terms 'plasma membrane' and 'plasmodesmata' are highly represented. Interestingly, 19 proteins out the 62 identified candidate proteins were found to belong to the FW2.2 co-immunoprecipitation proteome (Beauchet et al., 2024), and three of them crossed also with the refined PD proteome from Arabidopsis established by Brault et al. (2019), among which was Callose Synthase 10a (SICalS10a; Solyc03g111570) (Supplementary Table S3). It is also noteworthy that among the identified candidate proteins, was a subunit of the Cellulose Synthase complex (SICesA1; Solyc08g061100), which may relate to cell wall synthesis and cell expansion.

## Discussion

PLAC8 domain-containing proteins in plants belong to the FW2.2-LIKE/CELL NUMBER REGULATOR (FWL/CNR) protein family. Our understanding of the function of FWL/CNR proteins still remains poorly developed, but three distinctive functions have been identified so far
(Beauchet et al., 2021). These functions relate to (i) calcium uptake and signaling, (ii) metal ion transport and homeostasis, and (iii) organ size determination via the regulation of cell number. These three functions are not exclusive, as they may directly or indirectly influence each other, and examples of mutual actions on plant growth and metal ion transport (Song et al., 2015; Xiong et al., 2018; Li et al., 2019; Gao et al., 2020) or plant growth and calcium signaling (Rosa et al., 2017) have been reported. It is noteworthy that a function in improving resistance to a plant pathogen, namely Xanthomonas oryzae, has been described alongside to metal ion transport (Li et al., 2019).

So far, functional studies of *FWL/CNR* genes are still scarce in the literature. The first reports were dedicated to the functional characterization of *FW2.2* orthologs in important cereal crops such as maize and rice. In maize, the overexpression of *ZmCNR1* resulted in a reduction in overall plant size and organ size (namely tassel, ear and leaf size), thus affecting the plant biomass (Guo *et al.*, 2010). In rice, T-DNA insertion mutants of *OsFWL3* and *OsFWL5* displayed an increase in the grain weight and/or plant height (Xu *et al.*, 2013; Song *et al.*, 2015). Similarly, *OsFWL1/OsCNR1* (Ruan *et al.*, 2020) and *OsFWL4* (Gao *et al.*, 2020) were shown to act as negative regulators of rice grain width and weight, and tiller number and plant yield, respectively. In addition, the RNAi silencing of *GmFWL1* expression and the deletion of the conserved *PLAC8* domain of *GmFWL3* using CRISPR-Cas9 in soybean resulted in a significant reduction in nodule number in response to rhizobial infection, pointing out at a critical role for *GmFWL1* and *GmFWL3* in nodule organogenesis (Libault *et al.*, 2010; Cervantes-Pérez *et al.*, 2024). In all cases, these studies confirmed a role of certain *FWL/CNR* in regulating negatively cell number, thus influencing the organ size and/or development.

Due to the scarcity of data, we have undertaken in the present report a functional analysis of some *FWL/CNRs* in tomato, as to enrich our knowledge and to decipher their putative role in plant development.

**The *FWL/CNR* protein family presents a high diversity in protein characteristics, expression pattern and subcellular localization in tomato**

The *FWL/CNR* gene family in tomato comprises twenty-one members, showing a large diversity in protein length, ranging from 98 aa (SIFWL1) to 505 aa (SIFWL19). *FWL/CNR* proteins share as a common feature, the presence of the *PLAC8* domain made of two conserved motifs: the more or less divergent Cys-rich motif 1 of the type CLXXXXCPC or CCXXXXCPC, and the more conserved QXXRELK motif 2 (Figure 1). Since the CLXXXXCPC motif is present in FW2.2, *ZmCNR1*, *OsFWL1/OsCNR1* and *PfCNR1*, it would be tempting to address this motif as a
signature for FWL/CNR proteins involved in the regulation of organ growth. However, OsFWL4 and OsFWL5 which regulate rice grain weight, harbor the CCXXXXCPC motif. This latter motif has been described as important for conferring the Cd resistance function (Song et al., 2004). Interestingly, OsFWL4 and OsFWL5 both enhanced cadmium resistance too in yeast cells, and OsFWL4 acts as a transporter ensuring the translocation of Cd from roots to shoots in rice (Song et al., 2015; Xiong et al., 2018). Therefore, the sole nature of the PLAC8 Cys-rich motif does not indicate any function with certainty, and even less with the occurrence of different divergent motifs present in the tomato FWL/CNR family members. This was particularly obvious for the three SIFWL proteins we characterized functionally in deeper details, namely SIFWL2, SIFWL4 and SIFWL5, which harbor a CCXXXXCPC, ALXXXXFPC and AVXXXXLPC motif, respectively (Figure 1).

Despite clear levels of overexpression (Supplemental Figure S3), we did not observe any phenotypes in the 35S::SIFWL2, 35S::SIFWL4 and 35S::SIFWL5 lines (Figure 4). This suggests that constitutive overexpression likely fails to reproduce the native spatial and temporal regulation of these FWL/CNR genes, or may trigger compensatory mechanisms. On the contrary, the sole inactivation of SIFWL4 (CR-Slfw4 plants) and SIFWL5 (CR-Slfw5 plants) generated marked phenotypes during vegetative development: CR-Slfw4 lines produced longer roots (Figure 4B) and CR-Slfw5 lines showed reduced hypocotyl length (Figure 4C), and a spectacular reduction in leaf size (Figure 4D and 5B). Apart from SIFWL4 which seemed to be preferentially expressed in roots, these phenotypes could not be related with any specific gene expression: SIFWL2 was found more expressed in cotyledons, and SIFWL5 was expressed in all organs, without any preferential expression in leaves (Supplemental figure S1).

In the present study, we investigated the subcellular localization of eleven out of twenty SIFWLs (Figure 2). A diversity in subcellular localization was observed, as SIFWLs are located according to three distinct patterns: in both the cytosol and the nucleus for SIFWL7 and SIFWL8, at the ER for SIFWL1, -6, -9; -10, -11 and -15, and at the PM for SIFWL2, -4 and -5. Interestingly, a punctate localization in the plasma membrane was observed for SIFWL2 and -4, which may be consistent with a membrane microdomain localization, as demonstrated for GmFWL1 and GmFWL3 in soybean nodules (Qiao et al., 2017; Cervantes-Pérez et al., 2024). Therefore, this diversity in subcellular localization might probably be indicative of a diversity in protein function.

To the exception of SIFWL7 and SIFWL8, all the tested SIFWLs were thus found targeted to membranous compartments, most likely in connection with the presence of the hydrophobic PLAC8 domain or TM domains in the case of SIFWL15. From the original structural analysis of
AtPCR1 (Song *et al.*, 2004), it has long been accepted, and widely and systematically reported in the literature, that the PLAC8 domain in plant FWL/CNRs is composed of two hydrophobic segments (including the CLXXXXCPC or CCXXXXCPC motif), predicted to form transmembrane domains (for a review, see Beauchet *et al.*, 2021). However, the use of currently available tools for transmembrane topology prediction revealed that SIFWL2, SIFWL4 and SIFWL5 may not cross the PM in the absence of TM domains, but are likely to be anchored in the outer leaflet of the PM via their PLAC8 hydrophobic domain (**Supplemental Figure S2**), Such a topology would thus be very similar to that demonstrated experimentally for FW2.2 (Beauchet *et al.*, 2024).

## SIFWL5 regulates leaf size and hypocotyl growth in tomato via cell expansion

Unexpectedly, we could demonstrate that the leaf and hypocotyl growth phenotypes in *CR-Sifw5* lines are not due to impaired cell divisions, as it would be expected for a member of the CELL NUMBER REGULATOR protein family, but from an impairment in cell expansion, as cell size in all three lines was greatly reduced (**Figures 5E, 5F, 6B and 6C**). Interestingly, this reduction in cell size was accompanied by a significant down-regulation of some leaf- and hypocotyl-expressed *EXPANSIN A* genes (**Figures 5G and 6D**). The contribution of *EXPANSIN* gene expression in modulating leaf growth (Cho and Cosgrove, 2000; Goh *et al.*, 2012) and hypocotyl growth (Caderas *et al.*, 2000) is a long-standing observation. Especially for leaf growth, it is part of the complex molecular and hormonal network regulating leaf size (Gonzalez *et al.*, 2012).

Not all *EXPANSIN A* genes responded in the same way to *SIFWL5* loss-of-function. Indeed, among the genes specifically expressed in these two organs (Lu *et al.*, 2016), only *SIEXPA7*, *-10* and *-12*, and *SIEXPA3*, *-7* and *-18*, were down-regulated in leaf and hypocotyl, respectively (**Figures 5G and 6D**, and **Supplemental Figure S6**). The origin of this discrepancy is not known. It can be related to the specific function of *EXPANSINS* or timing and localization of *EXPANSIN A* gene expression. For instance, *SIEXPA2* is the major gene expressed in stems and hypocotyls, but its expression is maximal at the top of the hypocotyl, corresponding to the zone of rapid elongation, and very low in the middle part of the hypocotyl where cells have already expanded (Caderas *et al.*, 2000). Whether this discrepancy may relate to any modification in the hormonal regulation of *EXPANSIN A* genes following *SIFWL5* loss-of-function, is an intriguing matter of investigation. It was shown that *SIEXPA3*, *-7*, *-12*, and *-18* were all positively regulated by auxin, while *SIEXPA10* was positively regulated by GA (Lu *et al.*, 2016), two major hormones involved in the stimulation of cell elongation during plant growth.
Not only the inactivation of SIFWL5 affected leaf growth, but it altered also leaf morphology into simpler leaves, displaying a reduced number of the third primary leaflet, of secondary leaflets and intercalary leaflets (Figure 5C-D). This phenotype is fully relevant with hormonal control of compound leaf morphogenesis and differentiation in tomato (Bar and Ori, 2015). Indeed, gibberellin (GA) negatively regulates leaf complexity in tomato, as increased GA levels or GA signaling accelerate leaf maturation and induce simpler leaves than in wild type (Jasinski et al., 2008; Yanai et al., 2011). In addition, GA and cytokinin (CK) present a mutual antagonistic interaction during tomato leaf development (Fleishon et al., 2011). How to relate the molecular origin of the leaf phenotype in CR-SIfwl5 plants and phytohormone signaling remains to be established, but the following step in the functional characterization of SIFWL5 may provide new lines of research.

SIFWL5 is enriched at plasmodesmata, within a protein complex composed of plasmodesmata- and plasma membrane-specific proteins

We have shown that SIFWL5 is enriched at PD (Figure 3), where it probably participates in a protein complex composed of plasma membrane- and plasmodesmata-specific proteins (Figure 7). Interestingly, a significant number of candidate proteins that co-immunoprecipitated with SIFWL5 were found enriched at PD. This finding suggests that SIFWL5 may have a function in cell-to-cell communication mechanisms for the control of leaf and hypocotyl growth. SIFWL5 co-immunoprecipitates with Callose Synthase 10a (CalS10a; Solyc03g111370), which contributes to callose homeostasis at the cell plate and at PD, thereby regulating cytokinesis and the symplastic molecular exchanges between neighboring cells via the permeability of PD (Saatian et al. 2018). CalS10 is required for normal plant development, as silencing CalS10 results in retarded growth: plants display dwarfism, with smaller stem and smaller leaves (Töller et al. 2008), a phenotype originating from either impaired cell division or cell expansion. CR-SIfwl5 loss-of-function plants displayed a similar phenotype, i.e. a reduced hypocotyl length and leaf surface, due to impaired cell expansion. However, our data indicates that SIFWL5 does not control directly callose deposition dynamics via regulating the CalS activity, thereby modifying the PD aperture. Indeed, according to our immunolocalization assays the absence of a functional SIFWL5 was not associated with measurable differences in callose deposition in leaves (Supplemental Figure S8). Therefore, the mode of action of SIFWL5 at PD appears quite different from that of FW2.2 (Beauchet et al., 2024), and the putative interaction of SIFWL5 with CalS10 may only reflect its protein environment at PD.
Interestingly, SICesA1, a subunit of the Cellulose Synthase complex, was identified among the candidate proteins that co-immunoprecipitate with SIFWL5. Cellulose is the most abundant β-glucan polysaccharide of the plant cell wall, which self-assembles as microfibrils to strengthen the cell wall and contribute to the direction of cell growth during the cell expansion process (Schneider et al., 2016). Both cellulose and callose are synthesized at the PM by the large respective synthase complexes, cellulose synthases and callose synthases, and the formation of cellulose/callose networks has been demonstrated, especially as a layered cellulose/callose architecture in cell walls of epidermal leaf cells (Falter et al., 2015). Whether the SIFWL5 function in controlling leaf growth requires a regulatory role on Cellulose synthases during cell expansion remains an exciting matter of future investigation.

Unfortunately, we were unable to provide any direct experimental evidence for the physical interaction between SIFWL5 and CalS10 or SICesA1, identified as putative interactors from our IP-MS/MS proteomics assays (Figure 7 and Supplementary Table S4). First, the very large size and multi-transmembrane domain nature of CalS and CesA make them extremely challenging to clone and express in heterologous system. Second, both CalS and CesA proteins possess N- and C-terminal ends that face the cytosol (Verma and Hong, 2001; Kumar and Turner, 2015), whereas SIFWL5 displays both N- and C-terminal regions in the apoplast (Supplemental Fig. S2), alike its counterpart FW2.2 (Beauchet et al., 2024). Therefore, this differential orientation precludes proper reconstitution of split-ubiquitin fragments or fluorescent tags, even if fused, resulting in false negatives or structural artifacts when using classical interaction methods, such as Yeast-two-hybrid or split-ubiquitin. Moreover, plasmodesmata and plant-specific membrane microdomains have unique lipid composition and regulatory environments that do not exist in yeast. Consequently, assays in yeast may not reflect biologically relevant interactions in plants. While in planta approaches (such as FRET-FLIM/BiFC) might appear suitable, the opposite orientation of N- and C-termini and thus this difference in protein topology prevent reliable reconstitution, and compromise the demonstration of a direct structural interaction between SIFWL5 and CalS10 or SICesA1.

As key elements of the cell-to-cell communication machinery, PDs are implicated in processes guaranteeing the collaborative function of the cells, developmental and patterning events (Petit et al., 2020), enabling the intercellular trafficking of different mobile signaling molecules, such as small RNAs, metabolites, hormones and transcription factors (TFs) (Wu and Gallagher, 2012; Brunkard et al., 2013). Being localized at PD, SIFWL5 may harbor a regulatory role on plasmodesmatal activity. Hence, we can hypothesize that SIFWL5 function is to regulate the cell-to-cell movement of molecules/signals promoting leaf cell expansion and leaflet initiation.
Obviously, the identification of such signaling molecules belonging to transcriptional networks involved in pathways affecting cell growth (Davière and Achard, 2013), and leaf growth (Gonzalez et al., 2012; Bar and Ori, 2014), would represent a giant leap in understanding the SIFWL5 functional role at PD.

In conclusion, we are far from deciphering the functional complexity of the FWL/CNR protein family during plant and organ development. Unexpectedly, we have been able to demonstrate that SIFWL5 contributes to the regulation of cell expansion during organ growth, which has never been reported before for a member of the CELL NUMBER REGULATOR protein family. Therefore, this study may lead us to revise our perception of the FWL/CNR function in regulating organ size. Whatever the mechanism is involved, cell division or cell expansion control, what may really matter is the potential function in cell-to-cell communication as to regulate organ growth.

Acknowledgements

We express our deepest thanks to Isabelle Atienza, Aurélie Honoré and Valérie Rouyère, for taking care of the plant culture in the greenhouse. We thank Jonathan Dragdwidge (VIB-UGent for Plant Systems Biology, Ghent University, Belgium) for sharing the unpublished *pH3.3::SP-mTagBFP2-HDEL* and *pUBI10::mScarlet-1* plasmids. Mass spectrometry experiments were carried out using the facilities of the Montpellier Proteomics Platform (PPM, MSPP site BioCampus Montpellier), a member of the national Proteomics French Infrastructure (ProFI UAR 2048) supported by the French National Research Agency (ANR-24-INBS-0015, Investments for the future F2030). The microscopy analyses were performed in the Bordeaux Imaging Center, a service unit of the CNRS-INSERM and Bordeaux University, member of the national infrastructure France Biolmaging, and supported by the French National Research Agency (ANR-10-INBS-04), with the great help of Lysiane Brocard.

Author contributions

N.G., C.C. and N.B. conceived the project and designed the research. A.B., L.E., L.B. and N.B. performed the research. V.R. performed the IP-MS-MS proteomics experiments. All authors analyzed and discussed the results. A.B., N.G., C.C. and N.B. wrote the manuscript with input from the other authors.
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**Conflict of interest**

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The authors declare that they have no conflicts of interest.

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**Funding**

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This work was carried out with the financial support of the French Agence Nationale de la Recherche (grant no. ANR-20-CE20-0002), the GPR Bordeaux Plant Sciences in the framework of the IdEX Bordeaux University 'Investments for the Future' program, and the French Ministère de l'Enseignement Supérieur et de la Recherche (PhD grant to A. Beauchet).

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**Data availability**

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The Mass Spectrometry Proteomics data underlying this article have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2022) partner repository with the dataset identifier PXD061438.

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<table><caption>1 Table I. Proposed nomenclature of *Solanum lycopersicum* FWL genes.</caption><thead><tr><td>Gene ID</td><td>Proposed Name</td><td>Name from Thibivilliers et al. (2020)</td><td>Name from Ran et al. (2023)</td></tr></thead><tbody><tr><td>Solyc02g090730</td><td>FW2.2</td><td>FW2.2</td><td>SIFW2.2</td></tr><tr><td>Solyc05g009620</td><td>SIFWL1</td><td>SIFWL1</td><td>SIFWL9</td></tr><tr><td>Solyc01g005470</td><td>SIFWL2</td><td>SIFWL2</td><td>SIFWL1</td></tr><tr><td>Solyc04g007900</td><td>SIFWL3</td><td>SIFWL3</td><td>SIFWL8</td></tr><tr><td>Solyc03g119660</td><td>SIFWL4</td><td>SIFWL4</td><td>SIFWL6</td></tr><tr><td>Solyc06g066590</td><td>SIFWL5</td><td>SIFWL5</td><td>SIFWL13</td></tr><tr><td>Solyc03g120600</td><td>SIFWL6</td><td>SIFWL6</td><td>SIFWL7</td></tr><tr><td>Solyc08g013910</td><td>SIFWL7</td><td>SIFWL7</td><td>SIFWL14</td></tr><tr><td>Solyc08g013920</td><td>SIFWL8</td><td>SIFWL8</td><td>SIFWL15</td></tr><tr><td>Solyc10g081410</td><td>SIFWL9</td><td>SIFWL9</td><td>SIFWL18</td></tr><tr><td>Solyc12g013570</td><td>SIFWL10</td><td>SIFWL10</td><td>SIFWL20</td></tr><tr><td>Solyc12g037950</td><td>SIFWL11</td><td>SIFWL11</td><td>SIFWL21</td></tr><tr><td>Solyc02g083540</td><td>SIFWL12</td><td></td><td>SIFWL3</td></tr><tr><td>Solyc10g084260</td><td>SIFWL13</td><td></td><td>SIFWL19</td></tr><tr><td>Solyc09g007490</td><td>SIFWL14</td><td></td><td>SIFWL16</td></tr><tr><td>Solyc10g018920</td><td>SIFWL15</td><td></td><td>SIFWL17</td></tr><tr><td>Solyc05g051690</td><td>SIFWL16</td><td></td><td>SIFWL10</td></tr><tr><td>Solyc03g093200</td><td>SIFWL17</td><td></td><td>SIFWL5</td></tr><tr><td>Solyc02g079390</td><td>SIFWL18</td><td></td><td>SIFWL2</td></tr><tr><td>Solyc06g048790</td><td>SIFWL19</td><td></td><td>SIFWL11</td></tr><tr><td>Solyc06g048810</td><td>SIFWL20</td><td></td><td>SIFWL12</td></tr></tbody></table>

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# Figure legends

Fig. 1. Phylogenetic analysis and protein structure of SIFWLs. (A) Phylogenetic tree built using maximum likelihood method was generated using MEGA11 (Tamura *et al.*, 2021). Values for 1000 bootstrap replicates are shown on each branch. The schematic representation of the SIFWLs protein structure were deduced from computation analysis of protein primary sequences. Transmembrane domains were predicted using DeepTMHMM (https://dtu.biolib.com/DeepTMHMM) (Hallgren *et al.*, 2022). (B) Amino acid sequence comparison of the PLAC8 domain between SIFWLs and FW2.2, using Muscle alignment (MEGA11).

Fig. 2. Subcellular localization of SIFWLs fused to YFP in N. benthamiana leaf epidermal cells. (A) ER localization witnessed by the use of the ER-specific marker construct *pUBI::mTagBFP2-HDEL*. Scale bar = 10 µm. (B) Cytosol and nucleus localization witnessed by the use of the marker construct *pUBI::mScarlet-1*. Scale bar = 10 µm. (C) PM localization witnessed by the membrane specific dye FM4-64. Scale bar = 20 µm. Intensity plots delineated by the dashed lines are shown for each co-localization pattern.

Fig. 3. SIFWL5 is enriched at PD. (A) Confocal microscope observations of SIFWL2, SIFWL4 and SIFWL5 localization in tomato leaf cells from 35S::SIFWL2-YFP, 35S::SIFWL4-YFP, 35S::SIFWL5-YFP plants. Scale bar = 10 µm. Intensity plots delineated by the dashed lines are shown for each co-localization pattern. AB, aniline blue. (B) PD index for SIFWL2, SIFWL4 and SIFWL5 localization in leaf cells from 35S::SIFWL2-YFP, 35S::SIFWL4-YFP and 35S::SIFWL5-YFP plants. n > 20 ROIs from 5 images. Statistical analysis: Kruskal–Wallis test with post hoc Dunn multiple comparison test. ***P < 0.01.

Fig. 4. Phenotypic analysis of SIFWL2, SIFWL4 and SIFWL5 gain-of-function (referred to as 35S::SIFWL2, 35S::SIFWL4 and 35S::SIFWL5) and loss-of-function plants (referred to as CR-Sifw2, CR-Sifw4 and CR-Sifw5 compared to WT plants. (A) fruit weight (at red ripe stage); n > 40 fruits from 4 plants per line. (B) Root length (at 17 DAG); n > 10 roots per line. (C) Hypocotyl length (at 17 DAG); n > 10 hypocotyls per line. (D) Mature leaf surface; n > 10 leaves from 4 plants per line. Statistical analysis: Kruskal–Wallis test with post hoc Dunn multiple comparison test. *P* < 0.05; ***P* < 0.01; **P* < 0.001; ****P* < 0.0001.

Fig. 5. Phenotypic analysis of mature leaves from *CR-Sifw5* plants compared to that of WT. (A) Schematic representation of a mature leaf from wild-type 'AC' background defining the terminal leaflet (TL), the primary (PL), secondary (SL) and intercalary leaflets (IL) in their
sequential order. (B) Silhouettes of the sixth leaf of 8-week-old CR-Slfwl5 plants. (C) Analysis of leaf composition in CR-Slfwl5 plants, numbers indicate the percentage of leaves presenting the corresponding leaflet. Color code refers to (A). (D) Determination of the mean TL, PL and IL surface in WT and CR-Slfwl5 plants. n > 10 leaves from 4 plants per genotype (each dot represents 1 leaflet surface measurement). Statistical analysis: Kruskal-Wallis test with post hoc Dunn multiple comparison test. ***P <0.001; ****P <0.0001. (E) Leaf epidermal cells of WT and the three CR-Slfwl5 plants. Bars = 100 µm. (F) Cell size determination in the leaf epidermis of WT and the three CR-Slfwl5 plants. Statistical analysis: Kruskal-Wallis test with post hoc Dunn multiple comparison test. *P <0.05; ***P <0.001; *P <0.05; ****P <0.0001. n > 12 images measurement from leaves 5 and 6 of 5 independent plants for each line. (G) Expression of SIEXPA7, SIEXPA10 and SIEXPA12 in leaves 3-4 of six-week old WT and CR-Slfwl5 plants.

**Fig. 6.** Phenotypic analysis of hypocotyls from *CR-Slfwl5* plants compared to that of WT. (A) Illustration of hypocotyl length in 2-week old WT and *CR-Slfwl5* loss-of-function tomato plants. Bars = 10 cm. (B) Hypocotyl epidermal cells in 10 DAG WT and *CR-Slfwl5* seedlings. Bars = 200 µm. (C) Cell size determination in the hypocotyl epidermis of WT and *CR-Slfwl5* plants. Statistical analysis: Kruskal-Wallis test with post-hoc Dunn multiple comparison test. ***P <0.001; ****P <0.0001. n > 12 images measurement from the central part of the hypocotyl of 5 independent plants for each line. (D) Expression of *SIEXPA3*, *SIEXPA7* and *SIEXPA18* in hypocotyls of 10 DAG WT and *CR-Slfwl5* seedlings.

**Fig. 7.** SIFWL5 co-immunoprecipitates with several plasma-membrane and PD-localized proteins. (A) Dot plots showing enriched proteins in 35S::SIFWL5-YFP IP-MS/MS experiments in young leaves. Red dot indicates significantly enriched protein (based on a Student's t-test with Benjamini-Hochberg correction *P* <0.05 and an enrichment ratio > 2). (B) Gene ontology (GO) enrichment analysis of the identified proteins following SIFWL5 co-immunoprecipitation.
<figure><img src="image_2.png" /><figcaption>Figure 1
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<table><thead><tr><th></th><th>TL</th><th>PL-1</th><th>PL-2</th><th>PL-3</th><th>IL-1</th><th>IL-2</th><th>IL-3</th><th>SL-1</th><th>SL-2</th><th>SL-3</th><th>SL-4</th></tr></thead><tbody><tr><th>WT</th><td>100</td><td>100</td><td>100</td><td>96</td><td>84</td><td>100</td><td>80</td><td>92</td><td>8</td><td>100</td><td>68</td></tr><tr><th>CR-Slfwl5 #1</th><td>100</td><td>100</td><td>100</td><td>58</td><td>0</td><td>58</td><td>42</td><td>8</td><td>0</td><td>25</td><td>0</td></tr><tr><th>CR-Slfwl5 #2</th><td>100</td><td>100</td><td>100</td><td>71</td><td>21</td><td>100</td><td>36</td><td>71</td><td>7</td><td>57</td><td>14</td></tr><tr><th>CR-Slfwl5 #3</th><td>100</td><td>100</td><td>100</td><td>64</td><td>57</td><td>100</td><td>43</td><td>14</td><td>0</td><td>43</td><td>7</td></tr></tbody></table>

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